U.S. patent number 6,420,105 [Application Number 09/627,082] was granted by the patent office on 2002-07-16 for method for analyzing molecular expression or function in an intact single cell.
This patent grant is currently assigned to University of Kentucky Research Foundation. Invention is credited to Eric Blalock, Kuey-Chu Chen, Patrick Kaminker, Philip W. Landfield, Olivier Thibault.
United States Patent |
6,420,105 |
Landfield , et al. |
July 16, 2002 |
Method for analyzing molecular expression or function in an intact
single cell
Abstract
A method for obtaining molecules from a substantially intact
single cell is disclosed. The method differs from prior methods
that obtain a variable fraction of a cell's contents and therefore
cannot quantitatively estimate the number of molecules in the
cells. The present method comprises isolating and harvesting a
substantially intact single cell from its organ tissue comprising
the steps of subjecting a tissue mass to a dissociation method so
that the cells are dissociated from the tissue to expose cell
bodies or cell processes, contacting a dissociated cell with a
device capable of collecting the cell from the tissue substantially
intact, withdrawing device with the cell attached, and then
isolating or detecting the molecules in the single cell.
Inventors: |
Landfield; Philip W.
(Lexington, KY), Thibault; Olivier (Lexington, KY),
Blalock; Eric (Lexington, KY), Chen; Kuey-Chu
(Lexington, KY), Kaminker; Patrick (Berkeley, CA) |
Assignee: |
University of Kentucky Research
Foundation (Lexington, KY)
|
Family
ID: |
27386706 |
Appl.
No.: |
09/627,082 |
Filed: |
July 27, 2000 |
Current U.S.
Class: |
435/4; 435/1.1;
435/270; 435/6.16; 435/91.1; 435/91.2 |
Current CPC
Class: |
G01N
33/5005 (20130101) |
Current International
Class: |
G01N
33/50 (20060101); C12Q 001/00 () |
Field of
Search: |
;435/4,1.1,6,91.1,91.2,270 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Irani et al. (J of Immunological Methods (1991) 139:223-231).*
.
Thibault et al. (Current Opinion in Neurobiology (1995) 5:
382-387).* .
Monyer et al. (Current Opinion in Neurobiology (1995) 5: 382-387).*
.
Sucher et al. (Neuron(1995) 14: 1095-1100)..
|
Primary Examiner: Horlick; Kenneth R.
Assistant Examiner: Spiegler; Alexander H.
Attorney, Agent or Firm: McDermott, Will & Emery
Parent Case Text
CONTINUING DATA
The present application claims the benefit of priority to U.S.
Provisional application No. 60/148,563, filed Aug. 13, 1999; and
No. 60/157,849, filed Oct. 6, 1999. The contents of these
provisional applications are incorporated herein by reference in
their entirety.
Claims
We claim:
1. A method of isolating and harvesting a substantially intact
single cell from its organ tissue comprising the following steps:
(i) subjecting a tissue mass to a dissociation method so that the
cells are dissociated from the tissue to expose cell bodies or cell
processes; (ii) contacting a dissociated cell on its membrane with
a device capable of collecting the cell substantially intact from
said tissue; (iii) withdrawing said device with the cell attached;
and (iv) harvesting said cell, wherein said cell is isolated intact
from its organ tissue.
2. The method according to claim 1, wherein said harvesting step
comprises collecting the cell by drawing the cell into the lumen of
a second collection device.
3. The method according to claim 1, wherein the cells are partially
dissociated from the tissue.
4. The method according to claim 1, wherein said tissue is a part
of the central nervous system.
5. The method according to claim 4, wherein said tissue is
hippocampal tissue.
6. The method according to claim 5, wherein said tissue is a
partially dissocated hippocampal "zipper" slice.
7. The method according to claim 6, wherein said cell is a
neuron.
8. The method according to claim 1, wherein step (ii) comprises
patching a first glass pipette onto the cell, wherein a negative
pressure is maintained on the cell membrane through the glass
pipette, and a tight seal is formed between the cell membrane and
the pipette.
9. The method according to claim 1, wherein said device is a
suction pipette or a sharp dissecting needle.
10. A method for obtaining molecules from a single cell comprising:
(i) isolating a substantially intact single cell from its organ
tissue comprising the following steps: (a) subjecting a tissue mass
to a dissociation method so that the cells are dissociated from the
tissue to expose cell bodies or cell processes; (b) contacting a
dissociated cell on its membrane with a device capable of
collecting the cell substantially intact from said tissue; (c)
withdrawing said device with the cell attached; and (ii) isolating
or detecting said molecules in the single cell.
11. The method according to claim 10, wherein said molecule is
nucleic acid, proteins, lipids, carbohydrates or any other
biochemical substance present in the cell.
12. The method according to claim 11, wherein said nucleic acid is
mRNA.
13. The method according to claim 12, wherein said mRNA is detected
by amplification through polymerase chain reaction.
14. The method according to claim 10, wherein said molecule is
detected by hybridization, autoradiography, fluorescence, protein
ligand interaction, or immunocytochemistry.
15. The method according to claim 10, wherein said harvesting step
comprises collecting the cell by drawing the cell into the lumen of
a second collection pipette.
16. The method according to claim 10, wherein the cells are
partially dissociated from the tissue.
17. The method according to claim 10, wherein said tissue is a part
of the central nervous system.
18. The method according to claim 15, wherein said tissue is
hippocampal tissue.
19. The method according to claim 10, wherein said tissue is a
partially dissocated hippocampal "zipper" slice.
20. The method according to claim 18, wherein said cell is a
neuron.
21. The method according to claim 10, wherein the isolated cell is
said single cell is assayed for functional activity before or after
disrupting the membrane in step (ii).
22. The method according to claim 10, wherein step (ii) further
comprises dissecting, amputating or isolating a portion of the cell
and measuring the molecular content of said portion.
23. The method according to claim 10, wherein said single cell is
mixed with other single cells to obtain a plurality of single
cells.
24. The method according to claim 10, wherein step (i)(b) comprises
patching a first glass pipette onto the cell, wherein a negative
pressure is maintained on the cell membrane through the glass
pipette, and a tight seal is formed between the cell membrane and
the pipette.
25. The method according to claim 10, wherein said device is a
suction pipette or a sharp dissecting needle.
26. The method according to claim 10, wherein said detection is
carried out in a microarray system.
27. A method for measuring a molecule(s) from a single cell
comprising: (i) isolating and harvesting a substantially intact
single cell from its organ tissue comprising the following steps:
(a) subjecting a tissue mass to a dissociation method so that the
cells are dissociated from the tissue to expose cell bodies or cell
processes; (b) contacting a dissociated cell on its membrane with a
device capable of collecting the cell substantially intact from
said tissue; (c) withdrawing said device with the cell attached;
(d) harvesting said cell, wherein said cell is isolated intact from
its organ tissue; and (ii) measuring said molecule(s) in the single
cell.
28. The method according to claim 27, wherein said molecule is
nucleic acid, proteins, lipids, carbohydrates or any other
biochemical substance present in the cell.
29. The method according to claim 27, wherein said nucleic acid is
mRNA.
30. The method according to claim 27, wherein said harvesting step
comprises collecting the cell by drawing the cell into the lumen of
a second collection pipette.
31. The method according to claim 27, wherein the cells are
partially dissociated from the tissue.
32. The method according to claim 27, wherein said tissue is a part
of the central nervous system.
33. The method according to claim 32, wherein said tissue is
hippocampal tissue.
34. The method according to claim 27, wherein said tissue is a
partially dissociated hippocampal "zipper" slice.
35. The method according to claim 33, wherein said cell is a
neuron.
36. The method according to claim 27, wherein the isolated cell is
assayed for functional activity before or after disrupting the
membrane in step (ii).
37. The method according to claim 27, wherein a portion of the cell
is dissected, amputated or isolated to measure the molecular
content in the dissected portion of the cell.
38. The method according to claim 27, wherein said single cell is
mixed with other single cells to obtain a plurality of single
cells.
39. The method according to claim 27, wherein step (i)(b) comprises
patching a glass pipette onto the cell, wherein a negative pressure
is maintained on the cell membrane through the glass pipette, and a
tight seal is formed between the cell membrane and the pipette.
40. The method according to claim 27, wherein said device is a
suction pipette or a sharp dissecting needle.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present application is directed to a single cell preparation
method, or a brain cell preparation that provides near-optimal
conditions for the accurate quantitation of gene expression in the
single cell or neuron. The present invention also provides for a
method for studying the functional activity in the same single
cell. Also, the method of the present invention relates to the
collection of a single neuron, with its membrane and dendritic
processes substantially intact and with a full set of mRNA
transcripts.
2. Brief Description of the Related Art
With the remarkable explosion in gene expression "chip" technology
in recent years it appears that it will soon be possible, perhaps
routine, to measure the expression of hundreds or thousands of
genes simultaneously, under a wide variety of physiological and
pathological conditions. In the foreseeable future, this new
technology will clearly overwhelm our capacity to analyze the
functional implications of even a fraction of the gene expression
patterns that will be found. Taking the central nervous system
(CNS) as an example, eventually unraveling the complex linkages
between multiple gene expression and CNS function will likely
require studies of physiological properties in the same neuron in
which multiple gene expression is also assessed.
In turn, such studies will depend on the availability of highly
specialized preparations. An optimal CNS preparation for relating
gene expression to function in the same neuron should seemingly
have three major attributes: 1) the ability to perform extensive
functional studies on the same single neuron that is subsequently
collected for gene expression analysis; 2) the ability to collect
the full set of mRNA transcripts from that same neuron; and 3) the
ability to collect the neuron with most of its processes intact, in
order to preserve subcellular and dendritic mRNA distribution.
However, the main preparations commonly used in brain studies of
gene expression, including assays in homogenized tissues (RNAse
protection assays, Northerns), in situ hybridization, acutely
dissociated neurons, or electrophysiological recording with
extraction of cytoplasmic contents (e.g., in non-dissociated slices
or cultures), do not provide optimal conditions for parallel
measures of function and gene expression in the same neuron. In
particular, none of these approaches routinely allows for the
collection of the entire cell, including such specialized cells as
neurons with their dendritic processes intact. Without a full set
of mRNA transcripts, accurate estimates of specific mRNA content
are difficult to obtain, and specific dendritically-targeted mRNAs
are lost.
Gray et al. discloses a partially-dissociated hippocampal slice
(e.g., the "zipper slice" which gradually opens--or unzips--along
the cell body layers) in young guinea pigs to provide improved
accessibility to neurons for patch clamp pipettes. However, Gray et
al. does not disclose the method of the invention whereby a
substantially intact single cell is isolated whereby substantially
all of the mRNA is detected.
The invention brings together technology encompassing cutting edge
instrumentation for electrophysiology, confocal laser scanning
microscopy (CLSM), immunoautoradiography and histochemistry,
real-time detection of PCR kinetics and new DNA "chip" technology
(GeneChip Scanner and Analysis Suite), to optimize and extend the
invention.
While there is clearly growing recognition of the value of single
cell expression-function studies in the CNS, it seems less well
recognized that the preparations that are most widely available for
linking function to gene expression in single cells are
significantly limited in their usefulness for these purposes. For
example, the physiological recording methods in which cytoplasm is
extracted through a pipette, do not yield the full complement of
the cell's mRNA nor do they allow correlations with topographic
mRNA distribution. The proportion of total mRNA extracted varies so
greatly from one cell to another that there have been few attempts
to estimate the total amount of any mRNA species in a given neuron,
or even to estimate relative or semi-quantitative levels. The
observations based on this method to date have been generally
limited to all-or-none types of findings, regarding, for example,
whether or not a gene is expressed in a given cell, or in some
cases, whether its ratio of expression to other genes (e.g., for
different receptor subunits) is changed (e.g., Sudweeks and Twyman,
1995). In contrast, semi-quantitative or quantitative estimates of
the absolute amount of expression of a gene in a cell generally
requires collecting the entire complement of a neuron's mRNA
transcripts. This seems particularly critical for studies on
function-expression correlation in the same cells since the
physiological/pathological properties of a cell (e.g., density of
channels or receptors, developmental stage, biochemical phenotype
or pathological change) may well reflect total level of a gene's
expression rather than the ratio to another gene's expression,
which might also be altered.
Values obtained with the ratio normalizing approach required when
fractional cellular contents are extracted, can be substantially
affected by the additional error contributed by variability in the
"control" message to which the target message is normalized, by
different efficiencies (e.g., in PCR) between the two messages, or
by the very common occurrence in which the normalizing message is
also up- or down-regulated in tandem, either by the conditions
under investigation or by other conditions of the cell (e.g., size,
metabolic activity) that affect both messages. The latter may
sometimes provide a control for non-specific effects, but in more
cases is likely to wash out the absolute value of the target gene
signal with which the investigated function may be correlated.
Further, it is becoming clear that differential targeting and
distribution of mRNAs within the cell (e.g., dendrites or soma)
play critical roles in the CNS (Steward et al., 1998; Kuhl and
Shehel, 1998). The collection of mRNA by cytoplasmic extraction
loses this differential distribution.
Thus, without the ability to obtain the full complement of mRNA
with its topographic distribution intact, it will clearly be
difficult to estimate the total amount of a mRNA species in a
neuron. In turn, this will make it extremely difficult, in most
cases, to draw quantitative conclusions about the relations between
gene expression and physiological function in individual cells. It
should be noted that although the term "gene expression" is used
here as a short hand somewhat interchangeably with mRNA content,
this is only for purposes of simplicity and it is well recognized
that the two are not necessarily equivalent. In the present
application, we focus only on the quantitation of total mRNA
transcripts, but controls are of course required in many types of
experiment before concluding that mRNA content directly reflects
expression.
Conventional Methods for mRNA Expression Measurement in the
Brain
The main available preparations are: a) Homogenized Tissues:
Neuronal and glial heterogeneity generally prevents the accurate
assessment of gene/mRNA expression relative to specific cell types
or functions, even if small regions of brain are dissected,
homogenized, and analyzed by conventional methods (RPAs, Northerns)
b) In Situ Hybridization allows visualization of topographic mRNA
expression in single cells, but because the tissue is fixed, does
not usually permit functional measures (electrophysiology, optical
imaging) from the same cells or collection of the mRNA pool for
subsequent amplification; also, quantitative analysis (grain
counting) is performed by sampling on one section, lending some
error to the estimate of total mRNA; c) Acute Dissociation of brain
neurons disrupts the membrane and amputates processes. It is not
suitable for use with aged or even mature adult neurons as these
are often highly traumatized by the procedure (Thibault et al.,
1995a); in addition the loss of dendritic processes precludes
studies of topographic differences in gene expression or collection
of total mRNA; and d) Electrophysiological Whole Cell Recording
(e.g., slices or culture) is compatible with many types of
functional studies but generally collects mRNA by aspiration of
cytoplasmic content, which as noted, is highly variable from cell
to cell and prevents reliable collection of the full set of mRNA
transcripts or separate study of somal and dendritic compartments.
Further, the Whole-cell method dialyzes the cell's interior which
can dilute substances that modify physiological function.
Thus, despite the rapidly developing sophistication in measuring
multiple gene expression, the currently available preparations are
not well suited for careful physiological-expression correlation
studies in the same neurons or even for collecting total mRNA in a
single neuron. However, as noted above, the partially-dissociated,
or "zipper", brain slice (Gray et al., 1990), appears to have the
potential to be a nearly ideal preparation for such studies in
brain cells of mammals of any age range (Thibault et. al., 1995a;
Thibault and Landfield, 1996;Chen et al., 1998 and Preliminary
Data).
Function and Expression: Statistical Value of Single Cell
Correlations
It is becoming increasingly clear that there is considerable
variability in the expression responses of different neuron types
in the same brain region and even among different neurons of the
same type. For example, it has been found that different neuron
types and/or neurons of the same general phenotype can exhibit very
different quantitative or topographical (dendrites vs. soma)
patterns of distribution of the same mRNA species. One recent major
study concluded that there are no general rules for mRNA
localization that apply to all neuron types nor are there
neuron-type-specific mechanisms that invariably regulate mRNA
distribution (Paradies and Steward, 1997).
Thus, testing a hypothesis that some aspect of gene expression is
directly linked to a specific function will in many cases require
correlational analyses of the degrees of association across these
highly variable individual cellular patterns. Statistically,
individual-sample correlation of course provides a more rigorous
test than co-variance among group means, since the degrees of
freedom (df) across which a possible correlation can vary in a
study of, say, 20 neurons in which both a physiological process and
mRNA content are measured in response to a treatment in each
neuron, would be 19 [df=n-1(20-1)]. However, if functional and
expression values are obtained separately in different neuron
groups (e.g., one set of neurons for recording, and one for mRNA),
then the physiology-mRNA correlation can only vary around the
number of experimental conditions (group means) and the associated
degrees of freedom (e.g., treatment or no treatment). Therefore,
the many more df's generally found in an individual sample
correlation study allow for clear statistical inferences and
probabilistic statements on the amount of variance in one variable
that is accounted for by variance in the other. This is not
possible for associations involving few df's (e.g., typically
across group means) which are consequently more susceptible to
chance associations.
A more general problem in this regard is that most major treatments
or conditions (e.g., aging, seizures, intense synaptic stimulation,
lesions, drugs, hormones, neurodegenerative disease, developmental
stages, etc.) presumably activate a large number of genes. As the
new microarray techniques for simultaneously assessing thousands of
genes increasingly come on line, it will become extremely difficult
to determine which if the many observed changes in expression are
relevant to function without careful same-cell observations of both
function and expression. Thus, the application of function-gene
expression correlations in single cells (e.g., with large multiple
regression correlation matrices and appropriate controls for
performing many comparisons) may become one of the key first steps
in attempting to interpret widespread gene activation in relation
to function and in dealing with the vast quantities of data that
the field is on the threshold of obtaining.
Prior Studies on Electrophysiology and Single Cell RT-PCR
The vast majority of previous studies on electrophysiological
recording and RT-PCR in the same single neurons have, as noted,
addressed all-or-none or message ratio questions, usually related
to whether or not a cell expresses a specific mRNA, and, if so,
whether or not it also manifests a particular physiological
property. For example, the GABAA receptor is thought to be composed
of 5 subunits, but there are almost 20 known subunits and variants
that can form the GABAA receptor. In transfected cells, different
combinations can influence affinity, pharmacological modulation,
channel conductance and single channel kinetics (e.g., Porter et
al., 1992). However the actual subunit combinations that occur in
vivo are not known. Consequently, many single cell recording-PCR
studies have been used to determine which subunits are expressed in
which cells, and how these combinations affect function (Sudweeks
and Twyman, 1996).
Although several prior studies have attempted semi-quantitative
analyses, a recent report (Tkatch et al., 1998) indicated a
quantitative correlation between an electrophysiological function
(K.sup.+ channel A-currents) and a measure of gene expression for a
related subunit (mRNA for the Kv4.2 K.sup.+ channel) in individual
brain neurons. However, that study was performed in acutely
dissociated basal ganglia neurons and therefore could not collect
total mRNA. In addition, a few studies in peripheral or
invertebrate neurons have also quantitatively correlated
physiological function and gene expression (e.g. Baro et al.,
1997). But no one has accomplished the isolation of a single cell
neuron having complexes processes as in an embodiment of the
present invention.
There is clearly an overall paucity of CNS single cell studies of
electrophysiological function-gene expression correlations, very
likely because of the limited availability of preparations
compatible with quantitative analyses.
The Partially-Dissociated ("Zipper") Slice. The
partially-dissociated slice preparation (often termed the "zipper
slice" for its tendency to gradually open, or unzip, along the cell
body layers), was originally developed by Gray, Johnston and
colleagues (Gray et al., 1990) in young guinea pigs. The partial
dissociation (unzipping) procedure involves mild enzymatic exposure
to proteolytic enzymes and gentle "shaking" (FIG. 1). It provided
unparalleled access to brain neurons for small patch pipettes and
therefore yielded the high quality recordings needed for single
channel analyses, with very little disturbance of cell structure.
This adaptation incorporated somewhat shorter and lower enzyme
exposure and more gradual "unzipping" (Thibault et al., 1995a), and
required several months to optimize. Of particular importance was
that the yield of high resistance (>20 G.OMEGA.) recordings from
healthy neurons was equivalent from young adult, mid-aged and aged
rat slices (Thibault and Landfield, 1996). Thus, with this
preparation, we were able to carry out the first single channel
analyses in brain neurons of aged mammals, and found an
aging-related increase in the estimated membrane density of
available L-type voltage sensitive Ca.sup.2+ channels (VSCC)
(Thibault and Landfield, 1996) (FIGS. 2, 3).
Although each preparation has advantages for certain kinds of
electrophysiological studies, it was noted above that neither the
acutely dissociated cell preparation nor the non-dissociated slice
or culture preparation permits consistent collection of the entire
mRNA complement in a single cell. Aspiration of cytoplasmic
contents through a whole cell patch pipette, in either of these
preparations yields a varying and unknown fraction of the mRNA
content from cell to cell (e.g., significant mRNA is likely trapped
by organelles or the collapsing cell structure, and most dendritic
mRNA is probably trapped in collapsing dendrites).
Moreover, most current electrophysiological preparations are
extremely limited in their usefulness for applications to mature
adult, much less aged, animals. Cell cultures generally utilize
embryonic or postnatal neurons and acute dissociation techniques
are usually focused on juvenile animals. In fact, we found that
acute dissociation was so traumatic for aged rat brain cells that
almost none survived the dissociation procedure (Thibault et al.,
1995a). Even most non-dissociated slice studies are performed on
juvenile or very young adult animals (cf. reviews, Thibault et al.,
1995a; 1998a).
In the zipper slice however, the neuron that is being recorded with
a cell-attached pipette can be easily and gently extracted from the
slice, with nearly all of its processes intact simply by gradually
withdrawing the pipette while still maintaining negative pressure
on the cell body. The entire long apical dendritic tree slides
readily out of the slice still attached to the non-disrupted cell
body. Most of the basilar dendrites also appear to be intact (FIG.
4). Aged animal neurons are extracted as readily and as
non-traumatically as are young. Thus, not only is the zipper slice
particularly suitable for large scale single channel recording
studies, but it appears to be a neurobiological preparation that
can provide a fully intact neuron with its morphological structure
preserved, for analysis of gene expression. Although this
preparation has usually been used with hippocampus, there appears
to be no reason why the preparation would not work from essentially
any brain region, or with any tissue of an animal in an animal of
any age.
Another method that can yield semi-quantitative estimates of mRNA
expression in a single neuron that is largely topographically
intact is in situ hybridization. However, even this approach
generally yields only a sample of total mRNA (i.e., on the section
through the cell) and can only be related to functional measures of
the same cell with difficulty.
Limitations of the Zipper Slice
Although the zipper slice is presently extremely well suited for
ion channel and imaging studies (cf. below), its main limitation
appears to be its sub-optimal suitability for synaptic studies. The
weakening of synaptic and tissue connections that makes it so
ideally suited for cell extraction also results in variable
synaptic connections, and therefore the zipper slice yields
inconsistent results in synaptic studies. However, the present
invention overcomes this limitation and greatly expands the range
of functional studies for which the zipper slice is highly
valuable.
SUMMARY OF THE INVENTION
A method is described for isolating a single cell from its organ
tissue, usually a neuron from neural tissue, while causing minimal
disruption of the cell's processes and membrane. The method of the
invention comprises extracting that cell from the tissue mass,
washing and transferring the cell, and then collecting the entire
cell. In the case of neurons, the processes are substantially
intact.
The method of the invention further comprises determining the
presence or amount of nucleic acid that is present in the extracted
single cell. The inventive method comprises collecting the entire
cell into a small pipette or tube filled with solutions and
substances that facilitate the detection by amplification or
hybridization of the messenger ribonucleic acid (mRNA) transcripts.
The cell membrane is then disrupted and methods for amplification
and art-accepted measurement techniques for measurement of small
quantities of mRNA or deoxyribonucleic acid (DNA) are applied.
In a preferred example, a brain slice from an experimental animal
is placed in a perfusion chamber and kept alive by oxygenation and
artificial cerebrospinal fluid (ACF). The slice is then subjected
to mild enzymatic concentrations and is nicked (cut) in a way such
that gradual dissociation of the slice occurs along the cell body
layers. Gentle vibration enhances this dissociation.
When the dissociation has proceeded sufficiently to expose cell
bodies, one of the cells is "patched" onto a glass patch pipette
(electrode) using standard patch clamp recording procedures. These
procedures involve suction (negative pressure) in the pipette, and
result in a tightly formed seal between the cell membrane and the
tip of the pipette. This tight seal facilitates low noise
amplification and recording of the electrical activity of the
cell.
After the recording or other physiological monitoring session is
completed, the cell is then pulled out of the tissue slice with
most of its processes intact by maintaining the suction pressure of
the pipette (electrode) and withdrawing the pipette by use of its
micromanipulator controls. The cells remain attached to the pipette
tip and readily slide out of the tissue mass with this method. The
nerve cell on the tip of the pipette is optionally washed in clean
ACF to remove extraneous mRNA and it is then transferred to a
larger collection or harvesting pipette filled with an appropriate
reverse transcriptase (RT) solution. The membrane is then disrupted
osmotically to allow the RT process to begin. The contents of the
cell are then transferred again to a tube containing an appropriate
solution for amplification, for example, by polymerase chain
reaction (PCR). Other methods for amplifying mRNA or DNA would be
equivalent to RT-PCR for purposes of the invention.
These and other objects of the invention will be more fully
understood from the following description of the invention, the
referenced drawings attached hereto and the claims appended
hereto.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a schematic drawing of the hippocampal "zipper slice"
split along the major cell body layers. (From Gray et al.,
1990).
FIGS. 2A-2D show a zipper slice preparation for recording single
Ca.sup.2+ channels from CA1 neurons from a young (A) and aged (B)
hippocampal slices. (C) and (D) show five depolarizations to +10 mV
as well as the average current ensembles below. Ensembles were
created from 15 such depolarizations. (From Thibault and Landfield,
1996).
FIG. 3 shows that L-type channel density was obtained by estimating
N from maximum simultaneous openings and calculating patch area
from pipetted resistance. Channel density increased with aging
(p<0.001) (n=19-35/group). (From Thibault and Landfield,
1996).
FIGS. 4A-4D show harvesting cells with intact neurites. A) The
recording electrode is patched onto a CA1 pyramidal neuron and
electrophysiologic data is recorded. B) The cell is then pulled
from the slice using the recording electrode (note intact
dendrites). C) The cell is cleaned of tissue debris by placing it
at the inlet port of the perfusion chamber, where uncontaminated
bath solution flows into the chamber. D) The apical dendrite is
drawn into the collection pipette by capillary action. Following
this, position pressure applied to the recording pipette releases
the cell in the collection pipette. (From Blalock et al.,
unpublished).
FIG. 5 shows a summary of the steps involved in collection and
molecular analysis of a recorded cell. 1) Enzymatic treatment (see
methods) partially frees neurons from tissue, providing
unobstructed access of the recording pipette to the cell membrane;
2) The cell is gently puled free of the tissue and collected. 3)
The collected cell is transferred to a reverse transcriptase (RT)
solution; 4) Subdivided into aliquots; 5) Each aliquot can then be
amplified for specific messages.
FIG. 6 shows Ca.sup.2+ channel activity and .alpha..sub.1D subunit
mRNA expression are associated. Representative on-cell patch L-type
Ca.sup.2+ channel activity (left) and .alpha..sub.1D mRNA gels
(right) show the 3 levels of electrophysiologic activity and mRNA
expression (note a 202 bp .alpha..sub.1D band at 45.sup.th and
54.sup.th cycle #). Top. Low levels of channel activity are
associated with low levels of expression. Middle. Medium levels of
channel activity are associated with medium levels of expression.
Bottom. High levels or activity are associated with high levels of
expression. (From Chen et al., 1998).
FIG. 7 shows that correlation between channel activity and message
level is specific for the message of interest. Top. Relationship
between Ca.sup.2+ channel activity and Ca.sup.2+ channel
.alpha..sub.1D mRNA level across individual neurons. A significant
positive correlation (r.sub.s =0.64, p<0.05, Spearman's
nonparametric test) was found between L-type Ca.sup.2+ current and
.alpha..sub.1D mRNA level (estimated by SC RT-PCR) across
individual cells. On the vertical axis cells were ranked according
to mRNA level. On the horizontal axis the same cells were ranked
according to channel activity. Bottom. Lack of correlation between
Ca.sup.2+ channel activity and calmodulin (CaM) mRNA level. No
significant correlation (r.sub.s =0.07; n.s.; Spearman's
nonparametric test) was found between CaM mRNA level and L-type
Ca.sup.2+ channel activity in the same cells as Top. (From Chen et
al., 1998).
FIG. 8 shows the localization of the .alpha..sub.1D mRNA (in situ
hybridization, ISH) and protein (immunoautoradiography, IAR) in
adjacent hippocampal sections. Note the extensive overlap of signal
for mRNA and protein in the two sections, and the correspondence of
the relative intensities of ISH and IAR in dentate gyrus (DG) vs
CA1 and CA3.
FIGS. 9A-9B show confocal microscopic analysis of .alpha..sub.1D
immunoreactivity in 0.5 .mu.m optical sections from hippocampal
slice preparations. Panel A illustrates an example of a confocal
image of two distinct cells in CA3 (arrows). Images show punctate
immunoreactivity localized in the cell cytoplasm and membrane
regions. Frequency distribution (B) of single-cell fluorescence
intensity from 0.5 .mu.m optical sections. All measures were taken
from cells wholly contained within the thickness of the tissue
section, and were sampled from the optical section containing the
greatest staining area from the cell.
FIG. 10 shows capturing cells from the zipper preparation. A) Cell
soma and neurites are well exposed by enzymatic treatment and
Ca.sup.2+ channel activity is recorded. B) Post-recording the cell
is gently pulled away from the slice and C) collected with a
harvest pipette.
FIG. 11 shows Indo1 loaded CA1 neuron in an intact slice. Image
reconstructed from multiple transients. 30FIG. 12 shows examples of
Indo1-AM-loaded CA1 neurons in the "zipper" slice preparation.
FIG. 13 shows transection of the major dendritic process of a
hippocampal neuron in culture during simultaneous whole-cell patch
clamp recording of Ca.sup.2+ currents (Na.sup.+ and K.sup.+
currents blocked by TTX, TEA and Cs).
With the neuron's membranes potential voltage clamped at -70 mV,
transection of the large process did not cause disruption of the
recording configuration, or alter the whole-cell Ca.sup.2+
currents. Similar results have been obtained in many other neurons.
(From Thibault et al., 1995b).
DETAILED DESCRIPTION OF THE INVENTION
Many studies have examined RT-PCR or other methods of amplification
of mRNA or DNA contents of a single brain cell after recording
procedures (see review in Monyer and Lambolez, 1995; Sudweeks and
Twyman, 1996; and Mackler and Eberwine, 1993). However, all of
these prior studies have either extracted the internal contents of
the cell by aspiration, after breaking the membrane, or used acute
dissociation procedures to isolate the cells. Unlike the partial
dissociation method used in this application, the acute
dissociation procedures massively disrupt the cell's plasma
membrane and amputate most of its processes (Kay and Wong,
1982).
A significant aspect of the invention is the adaptation or
partially dissociated tissue (e.g., slices) for the new use of
gene/mRNA expression analyses as well as a method for cell transfer
and preparation that works to provide accurate estimates of the
relative numbers of an mRNA transcript type within a single
physiologically characterized brain cell.
The invention is directed to a method for extracting a single cell
from a complex intertwined biological tissue without massively
disrupting its structure and/or its plasma membrane. The major
advantage of this method for any molecular or biochemical analysis
of molecular contents of the cell is that the entire set of the
cell's mRNA transcripts or other molecular species is captured.
Conventional methods inevitably lose some of the contents during
aspiration or acute dissociation, because of leakage after membrane
disruption, collapse of the cell's structure and trapping of
contents, loss of large processes and other causes. This loss of
molecular contents is variable and unknown from cell to cell and
therefore all measures of contents obtained by these conventional
procedures must be normalized as ratios to other substances of the
cell. The other substances are usually very heterogenous (e.g.,
total RNA) or are potentially subject to different and variable
forms of regulation than the substance of interest (e.g., another
mRNA species). This technical problem adds great additional sources
of variation to single cell measures of mRNA collected by
conventional means, and therefore has been one major impediment to
detailed quantitative analyses in single neurons.
The method of the invention has the important advantage over
conventional methods in that it allows the collection and
amplification of the full cell contents of a molecular species and
therefore, provides the potential for more accurate measurement of
single cell molecular content. Moreover, many tissues are
heterogeneous and contain diverse cell types, which is particularly
true for the Central Nervous System (CNS), and therefore,
measurement from tissue homogenates can be confounded. The ability
to measure accurately the molecular contents of a single cell will
circumvent this problem of this confounding heterogeneity.
Further, a second recognized advantage of being able to measure
single cell contents is that it confers the ability to correlate
gene expression of a cell, for example, with the physiological
function or other phenotype of the same cell, thereby greatly
facilitating the analysis of functional consequences of gene
expression. With the rapid advent of new technologies for measuring
expression of thousands of genes at a time (e.g., DNA chip
technology) the ability to determine which are most closely
correlated with function and phenotype will be increasingly
important.
A third major advantage of the method of the invention is that it
captures the cell with its molecular contents still distributed
anatomically within the cell in a topographical pattern similar to
that found in vivo. This is because the cell structure has not
collapsed and the processes are intact. In neurons, for example, it
is increasingly recognized that protein synthesis can occur in
dendrites and that the mRNAs found in dendrites are different
quantitatively from those found in the soma (Steward et al, 1998).
Therefore, obtaining the entire neuron with dendritic processes
intact will greatly facilitate the detailed analysis of molecular
regulation in different regions of the cell, which will enhance the
understanding of the function of dendritic mRNA.
Although the method has been developed in the brain, and seems
particularly well suited for CNS studies, it is to be understood
that the invention is not limited to isolation of neurons. The
inventive method can be applied to the isolation of a single cell
from any tissue source, such as, kidney, endocrine glands, and so
on. Any use of a gentle partial dissociation procedure and capture
of a complex single cell with its processes substantially intact
and without membrane disruption, or disruption of the processes,
with the goal of more accurately quantifying the content and/or
distribution of a molecular species in a cell are encompassed by
the invention.
The invention has been developed initially and validated for mRNA
of specific genes, but the application of these procedures for
accurate measurement of any molecular species, including proteins,
lipids or other biochemical substances are encompassed by the
invention.
The invention also encompasses any variation in which a neuron or
other complex cell type was collected and then specific parts of
that cell were dissected, amputated or isolated, for purposes of
selectively measuring molecular contents in the dissected component
or fraction.
The present invention is also directed to a novel use of partially
dissociated tissues for collecting and measuring an entire neuron
largely intact and with its full set of molecular contents. The
present application also describes new methods for achieving this
by extracting and collecting an individual cell without
substantially disrupting its membrane. The present application
provides evidence and describes procedures showing that this use is
feasible and reliable. In particular, the present invention also
describes methods for dissecting dendritic parts of an extracted
neuron. These methods for extracting, washing and transferring a
cell have been validated in our laboratory by showing that RT-PCR
of the mRNA contents for a Ca.sup.2+ channel gene is highly
consistent across different cells and also correlates
semi-quantitatively with phenotypic expression of the appropriate
Ca.sup.2+ channel. Thus, the present inventors are the first to
propose and illustrate the collection of the entire contents of a
largely undisrupted cell from a complex tissue such as the CNS.
One aspect of the invention is to define optimal conditions of
enzymatic exposure (concentration, duration, oxygenation,
temperature, etc.) that allow single neuron extraction from
multiple brain areas with the least disturbance of mRNA content.
Single cell PCR and in situ hybridization will be run in parallel
to cross-validate the conclusions. Three to five Ca.sup.2+
-regulatory genes (mRNA) will be examined in adult and aged rats
and subsequently, mice.
Another aspect of the invention is to develop optimal methods for
examining mRNA and protein content in the same zipper slice
neurons, by taking alternate sections for in situ hybridization and
immunoautoradiography. In other studies, a single neuron will be
extracted from a zipper slice gently placed on a coated glass slide
and fixed and strained with antibodies. Two methods for studying
mRNA in the same cell will also be compared: in situ hybridization
or RT-PCR, in both rats and mice.
Another aspect of the invention is to extend the range of
functional studies for which the zipper slice can be utilized by
developing new techniques to "unzip" the slice after the functional
studies are performed rather than before, as is presently done. The
major advantage will be that functional studies can then be
performed before synaptic contacts are altered by even the mild
enzymatic exposure of the current method. Two methods will be
compared for finding and extracting the same recorded cell after
the partial dissociation procedures: 1) loading with Ca.sup.2+
-sensitive dye during recording (before partial dissociation), and
finding that same cell on the CLSM after partial dissociation; and
2) maintaining pipette contact with the cell after recording is
completed, during subsequent gentle dissociation in the recording
chamber. Once this technique is established in rats, are extended
directly to mouse slices.
Another aspect of the invention is to develop methods to separate
dendritic and somatic morphological compartments for independent
PCR analyses. Several methods of dissecting the apical dendrite
away from the soma and cutting it into separate pieces are
compared.
Still another aspect of the invention is to extend the single cell
RT-PCR approaches of the invention to new technologies for large
scale expression profiles by adapting and validating methods for
amplifying the mRNA pool of a single cell. We develop proportional
amplification of mRNA transcripts to a degree that will be
compatible with large scale expression profiling using both new
"DNA chip" microarray technology and a 96-well PCR format.
Technology such as real-time PCR, TaqMan.RTM., ABI PRISM 7700
Sequence Detection System, and laser-activated DNA microarray
reading techniques such as GeneChip Scanner, Workstation, may be
practiced with the invention.
Hippocampal mRNA by In Situ Hybridization and RPA
Recent studies in our laboratories perhaps provide an example of
the additional statistical power that single cell correlations can
lend to the process of scientific inference. We adapted the zipper
slice preparation and found clear evidence of an increase in the
average membrane density of L-type voltage-sensitive Ca.sup.2+
channels (VSCCs) in CA1 cells of aged rats relative to young-adult
or mid-aged rats (Thibault and Landfield, 1996; please see
Appendix). Subsequently, in a series of experiments with in situ
hybridization and ribonuclease protection assays (RPAs), the latter
in homogenized hippocampus, we found that mRNA content for the
.alpha..sub.1D pore-forming subunit of the L-type VSCC also
increased with age in rats (Herman et al., 1998; please see
Appendix). In addition, these studies suggested a possible rise in
the .alpha..sub.1C variant of the L-type subunit, but no change in
mRNA for the .beta..sub.1B subunit (Herman et al., 1998). These
mRNA data in vivo closely echoed similar data from hippocampal
cultures, which showed an increase in .alpha..sub.1D mRNA over age
in culture that paralleled an increase in L-VSCC (Porter et al.,
1997).
An increase in membrane density of available ion channels can arise
from increased synthesis of channels, but it can also reflect a new
availability of previously silent channels (e.g., activated by
intracellular signaling processes such as phosphorylation,
co-factors, etc.). Although the finding of increased .alpha..sub.1D
mRNA was suggestive that new synthesis was involved in our findings
on L-VSCC, the similar direction with aging of group mean values
for channels and for mRNA clearly did not provide sufficiently
strong evidence for the conclusion that the increased L-VSCC
probably depended on increased gene expression. Several factors can
of course compensate for an increase in mRNA without increasing net
protein content (e.g., more protein turnover, and protein content
does not necessarily equate with functional channels). Moreover,
the results showing changes in multiple L-VSCC subunit messages
(.alpha..sub.1D and .alpha..sub.1C) reemphasized the more general
problem that, even if the increase in L-VSCC did arise from new
expression and synthesis, it would not be possible to determine
whether this was likely due to increased expression of
.alpha..sub.1D or .alpha..sub.1C, or both, or perhaps to an altered
expression ratio of one of the .alpha. subunits to .beta..
Single Cell RT-PCR
As discussed in Background of the Invention, correlation analyses
in single cells not only provide stronger statistical evidence that
an observed mRNA-VSCC linkage is not coincidental, but in addition
can clarify which of the many altered messages may be most closely
correlated with the altered function. For these reasons, we
determined to direct a major effort toward the development of
techniques that would allow us to test the .alpha..sub.1D mRNA and
L-VSCC correlation, as well as other function-expression
correlations, in single neurons. To take advantage of our ability
to harvest the full complement of mRNA transcripts from neurons in
the zipper slice, we invested much effort and resources in the
development of highly accurate message amplification techniques of
specific mRNA species from single neurons.
Initially, two basic approaches were compared: the "linear
amplification" method (Eberwine et al., 1992) vs quantitative PCR
(Monyer and Lambolez, 1995). However, in several cases we were
interested in relatively rare messages (e.g., the .alpha..sub.1D,
the glucocorticoid receptor (GR)), and therefore we chose, after
initial comparisons, to focus on the PCR method (FIG. 5).
Our earlier work with ribonuclease protection assays (RPAs) and in
situ hybridization had required constructing multiple probes and
optimizing conditions for each of the specific messages in which we
were interested. Many of our studies have focused on the hypotheses
that brain aging and age-related neuropathology are associated with
altered Ca.sup.2+ homeostasis (Landfield et al., 1992; Thibault et
al., 1998a) or that glucocorticoids, and perhaps other steroids,
modulate brain aging by altering Ca.sup.2+ homoeostasis (Kerr et
al., 1989; Landfield et al., 1992). Consequently, many of the genes
in which we are interested are related to Ca.sup.2+ regulation or
steroid receptors. In our previous RPA and in situ studies, we
developed and validated primers and probes for 11 genes
(.alpha..sub.1D, .alpha..sub.1C, VSCC .beta. subunit, calmodulin,
calbindin D, cyclophilin A, GR, mineralocorticoid receptor (MR),
plasma membrane Ca.sup.2+ -ATPase (PMCA) isoforms 1 and 2 and
glyceraldehyde dehydrogenase (GAPDH), the latter as an abundant
control. All have been validated for RPAs in homogenized tissues
and in situ hybridization (please see Herman et al., 1998 and
Porter et al., 1997-for Methods). At this point, new message probes
can be developed and added to the repertoire fairly routinely.
As we moved into single cell work, however, the conditions for each
primer had to be optimized again for single cell PCR. Moreover, in
validating the single cell PCR techniques it was critical to
confirm three points: 1) the same message could be amplified with
relatively similar efficiency in different cells; 2) the relative
rank order of samples for the expression of a given message was
consistent in tests run on different fractions of those samples;
and 3) the estimates were proportional to the fraction of sample
loaded from a given cell.
After these points were confirmed and RT-PCR conditions were
optimized (e.g., two-step of PCR for some mRNAs, a single gel band
for each target gene, consistent amplification efficiency patterns,
etc.) we established standard procedures and criteria for assaying
a message in a single cell (FIG. 5). These are: to be included in
data sets, mRNAs must show efficiencies (rates of rise) within a
specified range; each sample must have a full kinetic analysis
across PCR cycles (to ensure that measures are taken from a cycle
on the exponential phase, rather than at saturation) and the same
cycle for all samples (cells) being compared in an experiment must
be run on the same gel.
Single Cell Correlations of Electrophysiology and Expression
The procedures outlined above enabled us recently to complete two
major studies comparing .alpha..sub.1D mRNA content with
electrophysiological analyses of L-VSCC in the same individual
neurons. As shown in FIG. 6, there appears to be a striking
correlation from one of these studies between number of L-type
channels in the patch and .alpha..sub.1D expression in the same
cell. FIG. 7 shows the semi-quantitative (rank order) correlation
from one of these studies between membrane density of L-VSCC and
.alpha..sub.1D PCR product in 11 adult/aged pyramidal neurons,
quantified on a phosphorimager at a PCR cycle in the exponential
rising phase of the amplification curve (all cells compared on the
same cycle on the same gel). (Non-parametric rank order correlation
statistics were used because the cells were highly variable,
particularly on electrophysiology (FIG. 6), and were not
distributed normally on either variable). FIG. 7 (bottom panel)
also shows that similar analyses for calmodulin gene expression
from the same cells found no indication of correlation.
Thus, although these single cell studies (Chen et al., 1998 and in
prep.; Blalock et al., in prep.) obviously do not prove a causal
link between .alpha..sub.1D gene expression and L-VSCC levels, they
provide much stronger statistical evidence for an association than
was available from our separate independent analyses in groups
(e.g., Thibault and Landfield, 1996; Porter et al., 1997; Herman et
al., 1998). Further, these apparently represent the first
semi-quantitative correlations between electrophysiology and
expression in non-disrupted brain neurons.
Moreover, it should be noted that, while these studies have been
extraordinarily demanding and time-consuming (e.g., each message,
each sample and each validation procedure requiring a separate gel
and several days), new real-time sequence detection analyzer (ABI
PRISM 7700-Perkin Elmer) can be used, which allows simultaneous PCR
amplifications in 96 wells and provides an on-screen view of the
PCR kinetics, efficiency and specific product. Thus, the
time-consuming preparation of gels will be circumvented and many
more samples can be run with greater sensitivity and accuracy of
quantification. This will clearly provide a major boost to the rate
at which these studies can be carried out.
Localization of .alpha..sub.1D VSCC mRNA and Protein in
Hippocampus: Immuno-autoradiography and Imunofluorescence
An important aspect of being able to assess function in the same
cells in which gene expression is quantified, of course, is the
ability to evaluate protein content of those cells. Consequently,
we have been working to develop cellular measures of protein
content in the same cells as those in which mRNA is evaluated. At
present, we can consistently take alternate serial sections for
mRNA (in situ hybridization) and protein measures
(immunoautoradiography). At least for the .alpha..sub.1D subunit,
there appears to be a reasonable correspondence. FIG. 8 illustrates
the localization of .alpha..sub.1D subunit mRNA and protein in
hippocampus. Note the high degree of overlap between signals
demonstrated using cRNA probes and .alpha..sub.1D antibody,
consistent with likely co-localization. Note also the
proportionally high expression of both .alpha..sub.1D mRNA and
immunoreactivity (ir) in the dentate gyrus. FIG. 9 demonstrates
localization and semi-quantitation of .alpha..sub.1D -ir in
individual neurons of a hippocampal slice preparation, visualized
using immunofluorescence. Single cells within the slice have
variable levels of .alpha..sub.1D -ir, suggesting differential
quantitative protein content in individual neurons.
There are multiple means of collecting a neuron once it has been
loosened from its tissue connections. Therefore, any method in
addition to the tight seal-forming pipette technique that is used
to draw a largely intact neuron or other single cell from partially
dissociated tissue in order to collect its essentially full set of
molecular contents for purposes of analyzing some molecular or
biochemical properties, would also be a variation on this
invention.
The invention is directed to molecularly analyzing the full
contents of a single neuron or other cell type that has not been
substantially disrupted during collection, such that analyses of
molecular/biochemical process will be more accurate. A general
method that can achieve this is to gradually and partially
dissociate the enveloping tissue, such that connections are
weakened sufficiently to allow the extraction and collection of the
single cell without the massive disruption of membrane and loss of
contents that occurs when presently available conventional methods
are used for collection of molecular contents from a single cell.
The example provided herein was of extraction and collection by a
patch pipette and a collection pipette. However, since the main
basis of the invention is the novel concept of collecting a largely
intact single cell for molecular/biochemical analyses of its full
contents, any method of extraction, including use of a larger
suction pipette, a sharp dissecting needle or any other instrument
capable of collecting the cell from partially dissociated tissues
without major cellular disruption, would be a variation of the
invention.
The single cell isolation method of the invention is applicable to
any tissue, preferably the brain region in animals of any age,
including, but not limited to, guinea pigs, rats, mice and
humans.
The following examples are offered by way of illustration of the
present invention, and not by way of limitation.
EXAMPLES
Basic `Zipper` Slice Procedures
Because the zipper slice procedures will be used for essentially
each Example, they are outlined briefly below. More targeted or
specialized methods/procedures are described under the individual
Examples.
Conventional Slice Methods. Slices will be prepared using standard
methods, as previously described (Thibault et al., 1995a). After
rapid removal of the hippocampi, a central section of each
hemisphere is transected perpendicular to its axis and mounted onto
the stage of a vibratome (Vibratome series 1000). Slices are cut
(300 .mu.m thick) in standard artificial cerebrospinal fluid (ACSF)
and transferred to a 35 mm dish using a fire-polished Pasteur
pipette. Standard ACSF consists of (in mM): 114 NaCl, 30
NaHCO.sub.3, 10 glucose, 2.5 KCl, 2 CaCl and 8 MgCl. The slices are
transferred to an interface-type chamber (35 mm dish containing 2.1
mg pronase per 3 ml of oxygenated ACSF), placed inside an incubator
at 31.5.degree. C. The dish is continuously perfused with moist
oxygen/CO.sub.2 gas mixture (95% O.sub.2 2/5%CO.sub.2).
Zipper procedure. The slices will be placed in a warm (31.5.degree.
C.) oxygenated 35 mm dish containing 2.1 mg of pronase per 3 ml of
ACSF. After approximately 30 minutes, the contents of the 35 mm
dish will be replaced with 1.6 mg thermolysin in 3 ml of warm
oxygenated ACSF. After 20 minutes in thermolysin, a slice is then
washed several times and transferred to an analyzer cup containing
Ca.sup.2+ -free ACSF (ACSF without Ca.sup.2+ and 2 mM EGTA) and
gently shaken. Periodic visualization of the slice on the stage of
a microscope (or the CLSM) allows evaluation of the progress of the
"unzipping" process. After cell bodies are visible they are then
placed in a recording chamber and patched onto for recording and
harvesting for mRNA analysis (FIG. 2).
Harvesting an intact cell. As noted, we have developed a reliable
technique for harvesting an intact neuron using patch pipettes,
micromanipulators and a collection pipette. Using a cell-attached
patch pipette, the cell of interest is first gently teased away
from the rest of the slice (FIG. 10). The high resistance seal
formed between the glass and the cell membrane is used to
physically pull the cell away from the slice. This cell is washed
with a continuous flow of clean ACSF before being harvested. A
second, larger pipette (collection pipette) is then lowered into
the recording chamber. The collection pipette is empty and thus
"draws" the cell by capillary action into its lumen. The capillary
action is controlled by a luer-lock system placed on the back end
of the collection pipette (FIGS. 4, 10). The contents of the
collection pipette are then analyzed by RT-PCR analysis for
estimates of expression of specific genes of interest.
Example 1
Optimal Conditions for mRNA Analysis
a. Rationale: Previous work by Gray et al. (1990) and by ourselves
has established that neurons are quite healthy and viable in the
zipper slice preparation. Moreover the close correlation of
.alpha..sub.1D mRNA with L-VSCC in individual cells (FIG. 7), which
generally agree with in vivo data from animals (Herman et al.,
1998), suggests that mRNA content is not much altered by our
standard zipper slice procedure.
However, it may well be that under some conditions of preparation
(e.g., last slice of the day) there is deterioration of
physiological function and/or mRNA content. If this preparation is
to be used widely, it will be important to obtain the baseline
parametric responses, and to determine the optimal procedures as
well as limitations on its use. Markers and criteria for health
should also be defined. Thus, the main factors that regulate the
zipper process are systematically varied (duration, enzyme
concentration, oxygenation and temperature) and mRNA content
assessed as a function of this variation. The working technical
hypothesis for this Example is that, for each factor, an optimal
range exists such that mRNA will most closely approximate values
seen without any dissociation procedures.
b. Design: The overall design of this Example will involve
sequential experiments to systematically vary each of four main
factors; each factor will be varied in turn while the others are
held constant. After an optimal value is found for one factor
(e.g., duration) that value will be used while other factors are
varied. Each factor will be studied initially in 5 young adult (3-5
mo old) and 5 aged (27 mo old) male rats (F344.times.BN
Hybrids).
The basic design involves incubation of slices from 30-120 min in
enzymatic solution. Slices will be collected for analysis before
incubation and every 30 min thereafter to 120 min, slices collected
at each incubation point will be placed in a holding chamber and
then collected from the holding chamber at 3 hr, 4 hr and 5 hr
after the start of the incubation.
Half of the slices at each time point will be processed for in situ
hybridization (ISH) and half will be placed in the recording
chamber for collection of individual cells for subsequent PCR
analyses. In both the in situ and the PCR studies, three mRNA
species will be analyzed simultaneously: .alpha..sub.1D
.alpha..sub.1C and calmodulin. Because the in situ analyses can be
compared to slices taken at the zero time (prior to any enzyme
exposure), the time course of changes in cellular mRNA content for
3 messages can be examined. The effects of varying enzyme exposure
as well as holding duration will be quantified (by grain counting
in ISH and PCR for single cells). It should also be emphasized that
single channel recording data will also be obtained for many of the
cells studied by PCR.
Thus, these studies will determine whether duration of enzyme
exposure or holding affects measures of mRNA, and also whether this
effect varies with age. If little effect on mRNA is found, this
will indicate relative resistance of mRNA content to these
procedures and permit considerable latitude in experimental design.
On the other hand, if mRNA is found to decline after some critical
period, then this information will be of great importance for
future studies with this preparation.
It should be emphasized that the RT-PCR analyses of single cells
cannot be compared to a zero time point, or perhaps to the 30 min
point because some minimum of enzymatic incubation is necessary to
unzip the slice and expose the neuronal somata. However, at each
time point thereafter, the PCR estimates can be compared to ISH
estimates at the same time points. Although the measurement scales
of these two methods will differ, the patterns of change over time
can be tested for their degree of correspondence.
After exposure duration is determined, an exposure point that is
sufficient for unzipping the slice, but causes the least alteration
in mRNA for any of the messages either adult or aged rat neurons,
will be used for examining the effects of varying enzyme
concentrations in 10 other rats (5 young, 5 aged).
Using a design similar to that above, slices will be cut and placed
in varying enzyme concentrations in 8 different combinations (e.g.,
low Pronase-low Thermolysin, low Pronase/high Thermolysin, high
Pronase/low Thermolysin, high Pronase/high Thermolysin, as well as
high or low Pronase/normal Thermolysin and high or low
Thermolysin/normal Pronase (high Pronase=1.5 g/l; low Pronase=0.33
g/l; normal Pronase=0.67 g/l; high Thermolysin=1.33 g/l; low
Thermolysin--0.25 g/l; normal Thermolysin=0.5 g/l). Under each
condition, slices will be collected for both ISH and PCR.
These studies will clarify much more systematically than our
original development studies the minimum amount of enzyme needed
for unzipping, and the maximum that can be tolerated without
altering mRNA levels. This is important since higher concentrations
permit more rapid unzipping and lower concentrations are presumably
less disruptive. The interactions of enzyme concentration and aging
will also be clarified by these studies.
A similar series will also be carried out in 10 other animals to
examine the effects of temperature and some studies will also test
the rate of mRNA decay with low oxygenation.
Although 5 animals are proposed for each condition, it is likely
that more will be needed because of failed experiments or
variability or subtlety of the results, or the need for some pilot
tests; therefore an extra 20 young adult and 10 aged animals are
requested (Totals: 35 young, 25 aged animals).
c. Methods: The ISH methods required for this Example are described
in Example 2 below, along with methods for concomitant analyses of
protein content in these same tissues. The methods for recording
and preparation of the zipper slice are described earlier (please
see above) (for further detailed description of our slice
preparation and recording protocols, please see Thibault et al.,
1995a; Thibault and Landfield, 1996).
Example 2
Concomitant Protein Analyses in Slices and Single Cells
a. Rationale: Although the expression of protein in a cell is not
always directly reflective of gene expression, in many cases it can
be a critical index of function that is relevant to gene
expression. Thus, as we develop more sophisticated approaches for
measuring gene expression in a single cell, it would clearly be
highly valuable to also obtain semi-quantitative estimates of
proteins in the same cell. Moreover, just as it is critical to
optimize and examine the limits of conditions for mRNA content
(Example 1), similar parametric studies are needed for protein
evaluations. The present Example is directed at conducting
immunoautoradiographic (IAR) and immunohistochemical (IHC) analyses
of .alpha..sub.1D protein content in the same slices and neurons as
will be analyzed for mRNA in the various conditions studied in
Example 1 above. In addition, a new technique will be developed for
plating a single cell that has been extracted from the slice, but
is still on the tip of the recording pipette (FIGS. 4, 10). The
cell will be lowered onto a polylysine-coated microscope slide and
plated with the use of positive pressure through the pipette. Once
plated, the single cell will be fixed, stained for protein content
by IHC and evaluated by CLSM. Same single cell will also be
evaluated by one of two methods to be compared: 1) subsequent
collection of the cell for RT-PCR analyses (e.g., as in Cheetham et
al., 1997); or 2) ISH of the fixed cell.
Even if both techniques for assessing mRNA eventually prove too
inconsistent or are unreliable, this new method for evaluating
protein in a single cell from a zipper slice will be a valuable
addition to our ability to assess function and gene expression, as
it will provide a means for testing the correlation between protein
content and physiological measures. In turn, these physiological
measures can be related back to mRNA expression (single cell PCR)
in other neurons.
Analysis of Protein in Single Neurons Extracted from the Zipper
Slice
This experiment will employ confocal microscopic analysis of
.alpha..sub.1D subunit protein expression in single rat and mouse
cells processed for electrophysiological analysis using the zipper
slice technique. Following electrophysiological recording, the
target neuron will be gently lifted from the slice preparation by
the recording pipette and repositioned over a polylysine coated
photo-etched coverslip. The cell will be transferred to the
coverslip by gentle positive pressure. The cell will be allowed to
adhere to the coverslip for 2 hours, at which point the coverslip
will be transferred to a 24 well culture plate and frozen at
-80.degree. C. until analysis.
Immunohistochemistical staining of tissue will be performed as
above. In each immunohistochemical run, 8-10 recorded cells from
each group (young and old animals) will be processed
simultaneously. Histological sections of whole hippocampal slices
from the same animals will be processed in parallel, to provide
positive controls for staining and to allow correlation of
single-cell data with whole tissue analysis. In each run, controls
will include single cells processed with: 1) omission of primary
antibody; 2) omission of secondary antibody; 3) preabsorption with
peptide/protein, if available.
Immunohistochemical data will be analyzed semi-quantitatively.
Briefly, fluorescence intensity will be integrated across the cell
through summation of values obtained in 0.5 .mu.m optical slices
(using confocal microscopy). Integrated intensity values will be
obtained for each of 8-10 cells/group. The relationship of staining
intensity (in fluorescence units) to Ca.sup.2+ channel density will
be evaluated by regression analysis, allowing correlation of
protein staining with Ca.sup.2+ currents both within and across age
groups.
Note that mRNA levels will also be assessed in these same cells,
either by single-cell RT-PCR or the FISH method for mRNA
quantitation. At present, these experiments are designed to
systematically assess the .alpha..sub.1D VSCC. To the extent
feasible in the available time, we will implement parallel sets of
analyses to assess expression of other target proteins. In
addition, we are currently attempting analysis of two or more
protein species in individual neurons using dual label
immunofluorescence methods. Should this technique prove reliable
and reproducible, we will include co-localization analysis of
.alpha..sub.1D (or alternative regulated proteins) and housekeeping
proteins (e.g., GAPDH).
It appears likely that relative fluorescence intensity measures
will allow suitable resolution of protein expression for
correlation with electrophysiological measures recorded in the same
neuron. However, should fluorescence intensity prove an unreliable
measure of protein levels, we will employ IAR analysis/grain
counting to assess single-cell expression in an analogous
fashion.
d. Anticipated Outcomes: We anticipate that serial section analysis
will provide accurate estimates of relative levels of mRNA and
protein expression in single neurons in hippocampal slices. The use
of both radioisotopic and fluorescent applications should provide
cross-methodological validation of the individual techniques and
thus reinforce the interpretation of the data derived from each
technique. It should be noted that non-radioactive ISH is generally
less sensitive than radioisotopic labeling. For abundant proteins,
detection by FISH should not be problematic; however, less abundant
mRNAs may present sensitivity problems that may require resolution
by additional amplification steps. If this is the case, we will
attempt alternative labeling protocols (e.g., biotin labeling with
or without biotinylated tyramide amplification (see Speel et al.,
1998; Speel et al., 1999; Raap, 1998; McKay et al., 1997).
The use of mouse tissue for hippocampal slice methods has been
discussed previously (see Example 1). The mouse .alpha..sub.1D
subunit has been cloned (Perez-Reyes et al., 1990), and a mouse
cDNA suitable for will be generated by RT-PCR by our laboratory
(see Herman et al., 1996). Further, given the overwhelming sequence
homology of rat and mouse .alpha..sub.1D mRNA (94%), it seems
likely that the polyclonal .alpha..sub.1D antibody can be used to
assess protein expression in mouse hippocampus.
Example 3
Techniques to "Unzip" the Slice After Functional Studies Rather
than Before
a. Rationale and Design: As noted, the zipper slice preparation
already provides an excellent preparation for a wide range of
electrophysiological (e.g., single ion channel) and imaging
studies. However, the utility of the zipper for synaptic studies is
limited substantially by the weakening of synaptic connections
during partial dissociation which may be one reason the preparation
is not more widely used. Therefore, to greatly extend its potential
usefulness for functional studies, this Example is directed at the
examination of two new approaches for unzipping the slice after
functional studies. In the first approach, individual cells in the
intact non-dissociated slice will be impaled with a sharp electrode
containing a Ca2.sup.+ -sensitive indicator (Indo-1). Indo-1
fluorescence will be used to label the cell so that it can be found
again after partial dissociation. Although many indicators should
work in these kinds of studies, it will be advantageous to uses a
ratiometric Ca.sup.2+ -sensitive dye during these early
developmental studies to determine rapidly whether any of our
procedures are inducing toxic elevations of Ca.sup.2+ (from influx
or internal stores). During this period, extensive physiological
measures will be acquired [e.g. after hyperpolarization (AHP),
synaptic input-output curve, Na.sup.+ spike threshold, LTP, etc.].
Immediately following these measures, the sharp electrode will be
withdrawn and the slice will be exposed to enzymes and "unzipped".
After the cell layer opens, that same cell will be identified on
the CLSM, recaptured with a cell-attached patch pipette and
harvested for mRNA analysis. The second approach will be developed
in parallel studies. In this approach, we will utilize the patch
clamp, rather than the sharp pipette configuration in the
non-dissociated slice and no indicator dye will be used. Once a
patch is formed, a wide range of physiological studies will be
carried out. Following these, contact will be maintained with the
cell (monitored by a high resistance seal) during the subsequent
exposure of the slice to enzymatic digestion. Gentle mechanical
"teasing" will be used to unmask the cell, which will then be
gently extracted and harvested as previously described. In both
approaches, the goal will be to perform functional synaptic studies
before exposure to enzyme and to then find and collect the same
cell after unzipping.
b. Methods, Approach #1: Electrophysiological Intracellular
Recording and Cell Loading. CA1 pyramidal neurons will be impaled
with sharp recording micro-pipettes. The electrode tips are
standardly back-filled with 14 mM Indo-1 in 150 mM Potassium
methylsulphate (KMeSO4) and 10 mM Hepes buffer (pH 7.2). The rest
of the electrode shank is back-filled with 2 M KMeSO4 in 40 mM
Tris-based buffer (pH 7.2). The pipettes generally manifest
resistances between 90-150 M.OMEGA. when filled with
indicator-containing solutions. The neurons consistently exhibit
input resistance (IR) between 50 and 100 M.OMEGA.. Current clamp
recording will be carried out with an Axoclamp 2A (Axon
Instruments) in bridge mode. Cells will be held at -70 mV with a
minimal holding current of 0.1-0.3 nA and Indo-1 allowed to diffuse
into a neuron over a period of .about.10 minutes, during which
baseline electrophysiological measurements (AHP, input resistance,
synaptic input/output (I/O) curves, Na.sup.+ spike threshold) will
be acquired. Synaptic activation is will be accomplished with a
bipolar stimulating electrode made from teflon-coated stainless
steel wires and placed into stratum radiatum (Schaffer-Commissural
pathway) approximately 200-300 .mu.m from the recorded cell.
LTP induction paradigm and electrode removal. Once cells have been
loaded with indicator and baseline electrophysiological measurement
have been taken, half will be subjected to a long-term potentiation
(LTP) protocol. In these, the synaptic pathway will be tetanized (5
sec at 50 Hz) at 150% of Na.sup.+ spike threshold. Ca.sup.2+
imaging measures and EPSP values will be acquired during this
protocol and at 5 minutes interval thereafter for the following 20
minutes (FIG. 11). At this point the sharp electrode will be
removed from the cell. This sharp pipette withdrawal technique is
not anticipated to be traumatic to the cell as the membrane is
known to reseal almost instantly, leaving behind sufficient
indicator to identify the cell at a later point. It should be noted
that this type of electrode removal approach has been used
successfully in a number of imaging studies (e.g. Tank et al.,
1988; Guthrie et al., 1991). In these experiments, cells were
loaded with indicator using sharp electrodes and the pipette was
then removed from the cell to permit better Ca.sup.2+ imaging by
closer apposition of the objective or even by "flipping" the slice
for study on an inverted microscope. In all of those studies,
measures of resting Ca.sup.2+ appeared to be within the normal
physiological range (.about.80 nM). Once the pipette is withdrawn
in our studies, the slice will then be transferred with a
fire-polished Pasteur pipette into a 35 mm dish to begin our
standard enzymatic zipper procedure. Subsequently, the slice will
be returned to the CLSM stage, and the indicator loaded neuron will
be identified (FIG. 12). That neuron will then be patched onto with
a cell-attached pipette (Axoclamp 2A, Axon Instruments) and
extracted from the slice (e.g., FIGS. 4, 10). Again, the advantage
of using cells loaded with a ratiometric indicator is that at any
point following the opening of the cell layer, the cell can be
monitored for toxic Ca.sup.2+ levels. Moreover, it is possible that
LTP-activated cells will show elevated Ca.sup.2+. Because no
recording will be performed at this stage, the seal quality does
not have to be excellent, which will improve our yield
considerably. Once collected, each cell would be assessed by RT-PCR
for three mRNAs. In an effort to improve on the preparation and to
minimize enzymatic exposure, we will determine the lowest
concentration of each of the enzymes necessary to find the loaded
cell, based in protocols proposed in Example 1.
These same studies will be carried out on hippocampal slices from
young-adult and aged animals (5-7 and 27-29 months old,
respectively) (40 animals per age group over two years). After
these procedures are systematized, we will also pursue the same
experiments in hippocampal preparations from C57BL/6J mice of
analogous ages. We propose to accomplish this by decreasing the
enzymatic concentrations and/or finding the right compromise
between cell accessibility and enzymatic digestion.
b. Methods, Approach #2: Maintaining Patch Contact
Throughout the Zipper Procedure. As an alternative to withdrawing
the pipette from the cell while unzipping of a slice, we propose to
also examine an alternative approach in which a cell would be
patched onto and recorded from before dissociation, but thereafter
patch contact would also be maintained during the enzymatic
procedure as well as throughout the gentle teasing of the tissue,
until the cell can eventually be extracted and collected for mRNA
analysis.
An intact, non-dissociated slice will be transferred onto the stage
of a conventional microscope equipped with electrophysiological
recording equipment and a perfusion chamber. Using either the
"blind" or the "visual" patch technique, a fire-polished recording
pipette will be used to patch on to the somatic membrane of a
neuron. Either the whole-cell or the cell-attached patch
configuration will be established, depending on the experimental
design. Standard whole-cell pipette solution consists of (in mM):
140 KMeSO.sub.4, 5 Hepes, 4 Tris ATP, 0.3 Tris GTP, 14
Tris-phosphocreatine and 0.1 Leupeptin. In current clamp mode,
measures of AHP amplitudes, EPSPs, I/O curves, and Na.sup.+ spike
thresholds will be determined using an Axopatch 200 amplifier.
After 10 min of baseline recording, an LTP protocol as described
above will be used to induce long-term physiological changes. Cell
electrophysiology will be monitored for an extra 20 minutes at
which time, enzymatic treatment of the slice will be started within
the perfusion chamber. After both sets of enzymes have been used
(approximately 45 minutes), the slice will be gently teased-apart
at the CA1 layer using glass prongs and fine micromanipulators to
unmask the recorded cell. We have already attempted this approach
several times and have successfully maintained input resistance of
a recorded cell throughout the enzymatic and separation phase of
the procedure.
For this approach, the slice must be held in place within the
recording chamber and whole cell patch clamping must be maintained
throughout the experiment. We currently hold the slice in place
using a modified stimulating electrode. This tool is a four-pronged
electrode array slightly curved to follow the curvature of the
Schaffer collaterals. It is used as a cleat to hold the slice in
place. Dorsally, will be embedded in the alveus of the hippocampal
slice and attached to an electronically-controlled micromanipulator
a fire-polished glass pipette. Teasing apart the slice will be
accomplished by gently pulling the tissue in a dorsal/ventral
direction using the glass pipette (thereby separating the cell
layer). Both the glass pipette and the stimulus cleat will be
positioned prior to patching and recording a cell. Cell harvesting
will be accomplished as described previously. These studies will be
conducted in parallel with those of the first approach outlined
above, and will overlap in time and use the same animals.
c. Anticipated Outcomes: It is anticipated that one or the other of
the above approaches will provide a successful method for
performing physiological studies prior to partial dissociation and
for the subsequent collection of the same cell after "unzipping".
This seems likely because there are no major theoretical obstacles
to these techniques, and the primary problems to overcome are those
that will simply require time-consuming, trial-and-error studies to
identify the most suitable methods. It is anticipated that after a
few months, it will become clear whether the first or the second
approach is the more promising, and we will then focus all
development efforts on that approach. All of the necessary
techniques are similar to those in routine use in our laboratories,
and we have available upright and inverted imaging microscopes, as
well as a range of patch clamp and current clamp amplifiers. Thus,
it appears highly probable that these studies will yield a set of
procedures, as well as electrophysiological criteria that will
provide a standard means of conducting physiological studies prior
to partial dissociation and single cell collection for RT-PCR.
Example 4
Separate Collection of Dendritic and Somatic Compartments for
Selective PCR Analyses
a. Rationale: Although the zipper slice procedure provides a means
of collecting a neuron with its processes intact, there are no
methods presently available for taking advantage of this intact
collection procedure and analyzing the anatomical compartments
separately. In this Example, we will develop methods for carefully
dissecting the different cellular compartments. These studies will
utilize the same animals, resources and schedule as in Example
3.
b. Methods: To control for the effects of transecting a cell into
two components, one subset of recorded neurons will be collected
intact as in the usual zipper preparation and analyzed for
.alpha..sub.1D mRNA. A second set of cells will have the soma
severed from the apical dendrite and both collected and processed
separately. We will then recombine the results for .alpha..sub.1D
mRNA from the severed components to determine whether cells that
have had their processes severed lose transcript, or whether the
sum of the two parts is, on average equivalent to values obtained
in non-transected cells. In further studies, cells will be recorded
from and extracted and the apical dendrite will be cut twice, once
at the soma and again, approximately 150 microns above the cell
soma. Each compartment will be analyzed by .alpha..sub.1D mRNA
content to determine whether the cutting process alters endogenous
mRNA levels. In a separate set of experiments, primers for mRNA
species that are known to be preferentially targeted to dendrites,
such as ARC, MAP2 and CAMKII.alpha. (Steward et al., 1998; Kuhl and
Skehel, 1998) will be developed and used for RT-PCR analyses of
gene expression. If the dissection process is accurate, these
species should be found at much higher levels in dendritic vs.
somatic fractions.
Cutting the processes. Our laboratory is highly experienced in
ablating single apical dendrites from cultured hippocampal neurons.
This can be done consistently while maintaining high resistance
patch recording (cf. FIG. 13). Here, we propose to compare cutting
with either 1) a glass pipette tool, 2) a sharp tungsten rod or 3)
the harvesting pipette. For both 1) and 2), after the cell is
extracted from the zipper slice, the dendrite will have to be
lowered gently onto a substrate to provide mechanical support for
transection. The bottom of the chamber would be used for this,
employing either a jagged glass tool or a sharp tungsten rod to
separate the two compartments. Each cell component would then be
collected using an individual collecting pipette. For 3) we propose
to follow our standard collection technique (cf. FIGS. 4, 10) but,
in this case, when the collecting pipette draws the apical
dendritic component of the recorded cell into its lumen, the
collecting pipette will be raised such that the dendrite entering
the harvesting pipette will be severed, a process that we will also
facilitate by cutting with a micromanipulator-mounted glass tool. A
second harvesting pipette will then collect the somatic component
remaining on the recording pipette.
c. Possible Outcomes: These studies will compare possible methods
for isolating separate compartments of a cell for independent gene
expression analyses. They will be performed in rats and mice and
should require about a year of trial-and-error experiments. At the
end, they will have generated standard protocols for isolating
single neuronal compartments for gene expression analyses. Most
laboratories that perform single cell recording should be able to
implement the protocols.
Example 5
Single Cell RNA Amplification and Gene Expression Profiling
a. Rationale: One of the goals for this invention is to develop
strategies for multiple gene expression analysis in single cells
and, in particular, to establish methods for profiling differential
gene expression in functionally characterized single neurons of
hippocampal tissues from various age groups of animals. As noted
above, the development of DNA microarrays or oligonucleotide chip
technology has provided a powerful tool for simultaneous
measurements of gene expression on a large-scale (reviewed in
Jordan, 1998). The techniques involve labeling a complex cellular
or tissue RNA sample and then using the probe to hybridize with
sets of specific gene targets arrayed on either high-density
membranes or solid glass support. Nevertheless, the techniques
necessary for analyzing the small amounts of starting RNA material
found in a single cell by these new methods are only beginning to
be developed. The main challenge of single cell mRNA analysis is
the generation of sufficient material for detecting specific mRNAs
in the very small RNA pool of a single cell. This requires
increasing the amount of labeled probes by amplifying the single
cell mRNA pool.
For single cell mRNA amplification, two approaches have been
developed: 1) Reverse transcription of cellular mRNA followed by
polymerase chain reaction (RT-PCR) (reviewed in Monyer and Jonas,
1995); and 2) Linear antisense RNA amplification (aRNA) using RNA
polymerase enzymes (Van Gelder et al., 1990; Eberwine et al.,
1992). Both methods have been used in numerous studies on gene
expression that analyzed individual single cells. The RT-PCR
approach is highly sensitive for detection and quantitation of
target mRNAs, providing that the primer sequences are known. The
aRNA amplification method is very useful for expression profiling
of multiple messages in single cells, and potentially can be
applied to profile a large number of genes with the new cDNA
microarray technology.
Kinetic single-target PCR methods were used for relative
quantitation of differential gene expression among single cells
(FIGS. 6, 7 and Chen et al., 1998). In our protocol, 1/5 of the
single-cell cDNA from RT is used for PCR of a target message.
Accordingly, up to 5 messages can be analyzed in a single cell.
Among those studied to date are several rare messages (i.e., the
L-type Ca.sup.2+ channel subunits .alpha..sub.1C, .alpha..sub.1D
and .beta..sub.1b), the Ca.sup.2+ binding protein, calmodulin (CM),
and an abundant "housekeeping" gene, glyceraldehyde-3-phosphate
dehydrogenase (GAPDH). Although kinetic single-target RT-PCR has
proven to be very sensitive and quantitative for our single-cell
expression/function studies, it has the drawback that only a few
genes can be analyzed per cell, under the current protocol.
The present invention expands the scope of these studies to
correlate functional measurements with the expression of a large
set of genes. The invention is directed to a protocol for single
cell mRNA amplification that is compatible with collection of
single neurons from zipper slices. The objectives will be to
generate sufficient material for: (A) Performing large-scale
quantitative kinetic PCR using gene-specific TaqMan probes for
differential expression assays of multiple genes (for example
multiple genes involved in cellular Ca.sup.2+ regulation and/or
Ca.sup.2+ signaling pathway) using a 96-well real-time PCR
instrument; and (B) Generation of fluorescent-labeled complex
sample probes that are sufficient to hybridize with cDNA
microarrays of expressed genes from a brain cDNA library.
b. Design: These studies will focus primarily on validating the
proportional amplification methods under development. This will be
accomplished by collecting multiple single neurons, and dividing
the cDNA from RT into halves. One half of the cell will be used to
estimate .alpha..sub.1D and CaM messages by our established
quantitative kinetics PCR techniques (FIG. 7) and the other half
will be amplified by the SMART PCR method (cf. below). After
amplification the amplified cDNA will also be analyzed for
expression of the same two messages (as well as others), by our
quantitative real-time PCR system, with gene specific TaqMan probes
(cf. below).
The validation of proportional amplification will be tested by
individual cell correlations between values obtained directly by
single target PCR and the values obtained following SMART PCR
amplification. Separate correlations will be run for .alpha..sub.1D
and CaM. If the amplification is reliable and proportional, then it
should be predicted that highly significant correlations will be
found. These tests will be performed in 15 young, 15 mid-aged and
15 aged rat neurons, to determine whether aging alters the
stability or accuracy of amplification. A mid-aged group will be
included in these studies to provide a careful assessment across
the full age range.
If the SMART PCR method proves unreliable, the same amplification
studies will be repeated with the aRNA method. Once a successful
amplification technique is developed for rats, we will then carry
out similar studies in young and aged mice. Following verification
of the methods in mice, we will examine large scale expression
profiling on Affymetrix cDNA microarray chips for mouse. The cDNA
databases will be searched and the mouse microarray (for example,
GeneChip Murine 19K subB Array) containing those Ca.sup.2+ relevant
cDNAs in which we are most interested will be tested in 5 separate
tests comparing young and aged neurons. Again, results will be
validated for selected genes by target-specific PCR and subsequent
correlation analyses between the two procedures across the same
cells.
c. Methods for Single-Cell mRNA Amplification: The hippocampal
"zipper" slice preparation will be used for the isolation of
individual live neurons, as described previously, and an entire
neuron will be harvested with a capillary microelectrode,
immediately placed into a chilled microcentrifuge tube containing
20U RNAse inhibitor in 5 ul solution, and processed for cDNA
synthesis. The cellular RNA of each single neuron is converted to
cDNA by reverse transcription using MMLV reverse transcriptase and
a synthetic oligonucleotide primer.
Two methods for single-cell mRNA amplification. In one established
procedure for single neuron linear aRNA amplification (Eberwine et
al., 1992), a synthetic primer containing oligo (dT).sub.15 and the
T7 RNA polymerase binding sequence attached at its 5'-end, i.e.
oligo(dT)-T7, is used in the RT reaction. The RT is followed by
second strand cDNA synthesis and the purified cDNA is then
transcribed with T7 RNA polymerase yielding amplified antisense RNA
pool. The aRNA can be labeled and used to hybridize with gene
specific cDNA clones by reverse Northern or Southern blotting. It
is estimated that one round of aRNA synthesis can generate about
2000-fold of the original RNA pool and usually two rounds of aRNA
synthesis are used for expression profiling, as carried out in
several studies (Eberwine et al., 1992; Mackler et al., 1992;
Mackler and Eberwine, 1993; Crino and Eberwine, 1996; Cheetham et
al., 1997; Nair et al., 1998; Callahan et al., 1998; Chow et al.,
1998). Although the aRNA amplification method is very useful for
single cell RNA analysis, it remains to be determined whether two
rounds of aRNA amplification can generate sufficient substrate for
detectable hybridization signals on new cDNA microarrays,
particularly for rare messages.
Very recently, however, an alternative PCR-based cDNA amplification
approach to generating an amplified cDNA pool was described (Endege
et al., 1999). In this recent report, Endege and colleagues
demonstrated that using the SMART PCR cDNA Synthesis Method
(CLONTECH) the high-, medium- and low-abundance transcripts in the
original RNA profile of biopsy samples could be amplified
representatively in the resulting cDNA. Moreover, the PCR amplified
cDNA can be used as complex probes for differential gene expression
profiling. This PCR cDNA amplification method uses a modified
oligo(dT) primer and a special designed 5' primer in reverse
transcription; during the single strand cDNA synthesis, the 5'
primer serves as an extended template at the 5' end of the RNAs to
allow continuation of transcription to the end of the primer by a
Switch Mechanism At 5' end of RNA Transcripts (SMART) of the RT
enzyme(CLONTECH). The resulting cDNA often contains the full-length
transcript with the 5'-primer sequence added at the end which then
serves as a universal priming site for subsequent PCR
amplification. Using 1 .mu.m of starting tissue RNA, approximately
1.25 to 1.75 mg of PCR amplified cDNA was obtained under optimized
conditions, i.e. optimal PCR cycles for maximum yield of full
length cDNAs (Endege et al., 1999). Moreover, as little as 50 ng of
the amplified cDNA was needed for generating complex probes for
expression profiling and maintaining sensitivity for low abundance
messages.
Either the aRNA amplification or SMART PCR cDNA synthesis methods
appear applicable for the single cell RNA amplification that is
necessary prior to large scale gene expression profiling. However,
since our current single-cell gene expression studies use PCR-based
single-target RT-PCR assays, we propose here to adapt the PCR cDNA
synthesis approach for our studies requiring RNA amplification of
single neurons. Individual neurons collected after functional
characterizations will be processed for cDNA synthesis and
amplification using the reagents from the SMART PCR cDNA synthesis
kit (CLONTECH). The manufacturer's instructions will be followed
with modifications adjusted for our single cell protocols, (i.e.
the entire cell will be used in each RT reaction using the 3' and
5' primers provided in the kit, followed by cDNA PCR amplification
using the primers and instructions in the manual). Optimal PCR
conditions, e.g. number of cycles, will be determined by monitoring
the kinetics of GAPDH cDNA synthesis as described in Endege et al.
(1999). As noted, to validate that the original expression profile
in a cell is proportionally represented in the PCR-amplified cDNA
using the SMART protocol, we will analyze in parallel the
expression of VSCC .alpha..sub.1D, .beta. and CaM messages before
and after amplification, using our established quantitative
kinetics PCR procedures (see Preliminary data) on subfractions of
cellular RNA samples.
Analysis of Multiple Gene Expression by Two Approaches: 1) 96-well
PCR; and 2) cDNA Microarrays
Approach #1: Differential expression of multiple messages in single
cells by large scale multiple single-target RT-PCR using gene
specific TagMan probes and quantitative real-time PCR. For
differential expression profiling of an intermediate-scale number
of genes, (e.g., a limited series of genes for cellular Ca.sup.2+
regulation and Ca.sup.2+ signaling), in single neurons, we will use
the real-time PCR system (ABI 7700 PE Biosystem, 96-well) for
quantitative single-target PCR, and will analyze in parallel
multiple messages in individual cells. RNA of a single cell will be
amplified by PCR cDNA synthesis (as described in the previous
section) and aliquots will be added to individual wells, each of
which will contain a message-specific TaqMan probe and primers for
gene-specific PCR. The basic steps of this approach are as
follows:
Single cell RNA amplification by PCR cDNA synthesis. In our current
single-cell RT-PCR protocol, the cDNA of a single cell is aliquoted
in amount suitable for analyzing up to 4-5 messages, including
several rare messages (<0.01% of the total RNA). These rare
messages such as VSCC subunits, require two-step PCR. As noted
above, to be able to analyze a larger number of messages from one
cell than we are capable of at present, will require amplification
of the single cell RNA pool. Thus, each single neuron collected in
the present studies will be used for RT, followed by PCR cDNA
amplification using the SMART PCR cDNA synthesis reagents
(CLONTECH) and modified procedures as described in the previous
section. However, if this procedure does not prove suitable, we
will use the aRNA amplification method. Although the latter may not
generate enough probe for the cDNA microarrays, it is almost
certain to be able to provide sufficient substrate for this
multiple single message PCR approach.
Multiple single-target PCR with gene specific TaqMan probes.
Aliquots of an amplified cDNA pool from a single cell will be
analyzed for the message level of multiple genes by quantitative
real-time PCR. In a 96-well format, up to 48 messages can be
analyzed with replicates in the adjacent wells. When running
parallel PCR on multiple genes, it is essential that each gene
specific probe and primers are selected for comparable PCR
conditions, i.e. similar GC% in the primer sequences so that a
similar annealing temperature can be used in a run. The selection
of primers is also crucial to ensure specificity for the target
message and to avoid complementary sequences within and among
primers. The optimal PCR conditions for a set of primers and probes
and its target specificity will be determined prior to performing
multiple PCR.
These methods could be used to investigate multiple gene expression
with aging, in an experimental scheme of using a 96-well format to
compare expression profile of a set of genes among cells from 3
different age groups (e.g., young, mid-aged and aged). Aliquots of
amplified cDNA of a cell will be added to each of 32 wells, each of
which contained a message-specific TaqMan probe and primers placed
in replicates between adjacent wells. The 16 messages to be
analyzed in parallel will be grouped for comparable PCR conditions
so that the reactions can be run at the optimal settings, e.g. the
annealing temperature, cycle numbers, etc. The same design will be
repeated 15 times for a set of three cells from three animals
(e.g., one of each age group) at a time, so that data eventually
can be obtained on 15 cells from each group for statistical
analysis.
Alternatively, we will also perform experiments using a scheme in
which one message is assessed at a time in multiple cells from each
age group. For example, using a 96-well format, 15 cells of each
age group will be analyzed in duplicates, plus the control samples
for a single message. In this scheme, sufficient data can be
obtained for statistical analysis of differential expression for a
specific message on each run.
Comparative real-time PCR quantitation. The use of
fluorescent-labeled TaqMan probes allows real-time measurements of
the accumulation of target-specific PCR products during an
ampliication process. Quantitation by TaqMan real-time PCR is
obtained by monitoring the accumulation of fluorescence signals
during PCR. The real-time PCR device (PE Biosystems ABI 7700) makes
it possible to obtain large-scale PCR kinetics on 96 individual
reactions simultaneously. Data processing is performed by the
software of the 7700 system and calculation of the initial amount
of a target message using a standard curve can be obtained.
Approach #2: cDNA microarray hybridization and expression
profiling. For large-scale gene expression profiling, the current
protocols for cDNA microarray hybridizations require labeled
complex cellular cDNA probes in the nanogram to microgram range,
even in conditions using highly sensitive two-fluorescence labeling
(Schena, 1995). To develop a protocol for microarray expression
profiling in single neurons, our initial experiments will determine
whether sufficient probe can be generated from a single neuron
using the SMART PCR cDNA amplification procedure described above.
If this proves too erratic or variable, we will then also examine
the aRNA amplification method. Only single neurons collected from
mouse hippocampal preparations will be tested in the microarray
studies, as the mouse cDNA GeneChip arrays are currently
commercially available (Affymetrix). The basic procedures for this
approach are outlined below:
Fluorescent probe synthesis. In a gene microarray hybridization
scheme, the labeled probe comprises the complex cDNA copies
obtained from cellular mRNAs. The complex probe is then used to
hybridize with cDNA clones of many target genes arrayed on a solid
support (Ramsay, 1998). Thus, signal production from each clone in
an array is governed by the abundance of individual message present
in the complex probe (reviewed in Jordan, 1998; Duggan et al.,
1999). For microarray assays of single cell RNA, the mRNA pool, as
noted, must first be amplified proportionally either by linear aRNA
amplification or the SMART PCR cDNA synthesis procedure. In the
proposed studies, the amplified cDNA or aRNA will be used to
generate fluorescent-labeled complex probes for hybridization with
murine cDNA library microarrays.
To generate a complex cDNA probe, we will use fluorescent labeling
and the two-color fluorescent probe hybridization procedure (Schena
et al., 1996) to assess differential expression in RNA samples from
at least two different age groups. The amplified cDNA or aRNA of a
cell will be labeled using a fluorescence-tagged nucleotide analog,
i.e. Cy3- or Cy5-dCTP (Heller et al., 1997) added in the RT or in
the RT random priming (Stratagene) procedure. Two fluorophores will
be used to independently label samples of two age groups. The two
probes will be mixed in equivalent concentrations, and hybridized
simultaneously to a microarray derived from a hippocampal or brain
cDNA library. To reduce cost at this stage of technical
development, the microarray that is commercially available
(Affymetrix) can be used. A control yeast mRNA, e.g. trp4 gene for
tryptophan biosynthesis, will be added at a known dilution
concentration in each RT labeling reaction to provide an internal
standard for calibration (Schena et al., 1995).
Probe hybridization and quantitation. The use of glass slide
microarrays requires a very small hybridization volume and a
resulting high probe concentration in the reaction mixture, thus
providing high sensitivity of the assay (Schena et al., 1995). At
this stage of technical development, the GeneChip murine cDNA
arrays (Affymetrix) can be used for hybridization of mouse cellular
complex probes. The probe hybridization and detection will be
carried out following the manufacturer's protocols
(Affymetrix).
Data Analysis. Analysis of cDNA microarrays will depend on laser
scanning activation of fluorescence with complex software packages
for detecting differential signals above background noise (e.g.,
Hewlett-Packard GeneScan and Workstation with Analysis Suite,
Affymetrix).
Utility
The most apparent use is for research purposes. Because of the
advantages outlined above, research on gene and other molecular
regulation, on phenotypic consequences of gene activity and on drug
discovery, among many others, could be enhanced.
However, because the method promises to provide more accurate
quantitative estimates of molecular species in a clearly identified
cell type, it is also possible that the method will be advantageous
for clinical diagnostic purposes, for example, in detecting genetic
or biochemical defects, tissue compatibilities for transplants,
identifying candidates for therapeutic regimens, and many other
possibilities.
In addition, the method could eventually be useful in therapeutic
approaches, by allowing the accurate characterization of
subcellular function in specific individuals, thereby allowing more
accurate selection of treatment or more refined implementation of
gene transfer, pharmacological or other treatments, among other
possibilities.
Whereas particular embodiments of this invention have been
described above for purposes of illustration, it will be evident to
those persons skilled in the art that numerous variations of the
details of the present invention may be made without departing from
the invention as defined in the appended claims. All of the
references cited herein are incorporated by reference in their
entirety.
REFERENCES Baro D J, Levini R M, Kim M T, Willms A R, Lanning C C,
Rodriguez H E, Harris-Warrick R M. Quantitative single-cell-reverse
transcription-PCR demonstrates that A-current magnitude varies as a
linear function of ain shal gene expression in identified
stomatogastric neurons. J Neurosci 17:6597-6610, 1997. Blalock E M,
Chen K-C, Landfield P W, Slevin J T. Correlated changes in L-type
Ca.sup.2+ channel activity and .alpha..sub.1D ain mRNA levels in
individual neurons of kindled rats. (In Preparation) Bowers G,
Cullinan W E, Herman J P. Region-specific regulation of glutamic
acid decarboxylase (GAD) mRNA expression in central stress
circuits. J Neurosci 18:5938-47, 1998. Callahan L M, Chow N,
Cheetham J E, Cox C, Coleman P D. Analysis of message expression in
single neurons of Alzheimer's disease brain. Neurobiol Aging
19:99-105, 1998. Cheetham J E, Coleman P D, Chow N. Isolation of
single immunohistochemically identified whole neuronal cell bodies
from post-mortem human brain for simultaneous analysis of multiple
gene expression. J Neurosci. Methods 77:43-48; 1997. Chen K-C,
Kaminker P G, Blalock E M, Thibault O, Landfield P W. Single-cell
RT-PCR and single Ca.sup.2+ channel recording in adult and aged
"zipper" hippocampal slices. Soc Neurosci Abs 24:1972, 1998. Chen
K-C, Blalock E M, Landfield P W. Correlation of L-type Ca.sup.2+
current amplitude with the .alpha..sub.1D mRNA level in single
neurons from hippocampal zipper slice preparation. (In
preparation). Chiang L W. Detection of gene expression in single
neurons by patch-clamp and single-cell reverse transcriptase
polymerase chain reaction. J Chromatogr 806:209-218, 1998. Chow N,
Cox C, Callahan L M, Weimer J M, Guo L, Coleman P D. Expression
profiles of multiples genes in single neurons of Alzheimer's
disease. Proc Natl Acad Sci 95:9620-9625, 1998. Crino P B, Eberwine
J. Molecular characterization of the dendritic growth cone:
regulated mRNA transport and local protein synthesis. Neuron
17:1173-1187, 1996. Cullinan W E, Herman J P, Watson S J. Ventral
subicular interaction with the hypothalamic paraventricular
nucleus: Evidence for a relay in the bed nucleus of the stria
terminalis. J Comp Neurol 332:1-20, 1993. Davis W P, Janssen Y M,
Mossman B T, Taatjes D J. Simultaneous triple fluorescence
detection of mRNA localization, nuclear DNA, and apoptosis in
cultured cells using confocal scanning laser microscopy. Histochem
Cell Biol 108: 307-11,1997. Dirks R W, Raap A K.
Cell-cycle-dependent gene expression studied by two-colour
fluorescent detection of a mRNA and histone mRNA. Histochem Cell
Biol 104: 391-5, 1995. Duggan D G, Bittner M, Chen Y, Meltzer P,
Trent J M. Expression profiling using cDNA microarrays. Nat Genet
Supplement 21:10-14, 1999. Eberwine J, Yeh H, Miyashiro K, Cao Y,
Nair S, Finnell R, Zeftel M, Coleman P. Analysis of gene expression
in single live neurons. Proc Natl Acad Sci 89:3010-3014, 1992.
Endege W O, Steinmann K E, Boardman L A, Thibodeau S N, Schlegel R.
Representative cDNA libraries and their utility in gene expression
profiling. BioTechniques 26:542-550, 1999. Giese K P, Fedorov N B,
Filipkowski R K, Silva A J. Autophosphorylation at Thr286 of the
alpha calcium-calmodulin kinase II in LTP and learning. Science
279:870-873, 1998. Gray R, Fisher R, Spruston N, Johnston D.
Acutely exposed hippocampal neurons: A preparation for patch
clamping neurons from adult hippocampal slices. In Preparations of
Vertebrate Central Nervous System In Vitro, Ed. H Jahnsen 324,
1990. Guthrie P B, Segal M, Kater S B. Independent regulation of
calcium revealed by imaging dendritic spines. Nature 354:76-80,
1991. Heller R N, Schena M, Chai A, Shalon D, Bedilion T, Gilmore
J, Woolley D E, Davis R W. Discovery and analysis of inflammatory
disease-related genes using cDNA microarrays. Proc Natl Acad Sci
USA 94: 2150-2155, 1997. Herman J P, Kwak S P, Watson S J.
Hybridization approaches to the study of adrenocorticosteroid
receptors in the CNS. In dekloet ERSutanto W (Eds.), Neurobiology
of Steroids, Academic Press Orlando pp. 189-210, 1994. Herman J P,
Dolgas C M, Marcinek R, Langub M C Jr. Expression and
glucocorticoid regulation of natriuretic peptide clearance receptor
(NPR-C) mRNA in rat brain and choroid plexus. J Chem Neuroanat; 11:
257-65, 1996. Herman J P, Morrison D G. Immunoautoradiographic and
in situ hybridization analysis of corticotropin-releasing hormone
biosynthesis in the hypothalamis paraventricular nucleus. J Chem
Neuroanat 11: 49-56, 1996. Herman J P, Spencer R. Regulation of
hippocampal glucocorticoid receptor gene transcription and protein
expression in vivo. J Neurosci 18: 7462-73, 1998. Herman J P, Chen
K-C, Booze R M, Landfield P W. Upregulation of .alpha..sub.1D Ca2+
channel subunit mRNA expression in the hippocampus of aged F344
rats. Neurobiol Aging 19: 581-587, 1998. Jonas P, Racca C, Sakmann
B, Seeburg P H, Monyer. Differences in Ca.sup.2+ permeability of
AMPA-type glutamate receptor channels in neocortical neurons caused
by differential GluR-B subunit expression. Neuron 12:1281-1289,
1994. Johnston A R, Black C, Fraser j, MacLeod N. Scrapie infection
alters the membrane and synaptic properties of mouse hippocampal
CA1 pyramidal neurones. J Physiol 1:500 (Pt 1): 1-15, 1997. Jordan
B R. Large-scale expression measurement by hybridization methods:
from high-density membranes to "DNA chips". J Biochem 124:251-258;
1998. Kerr D S, Campbell L W, Hao S-Y, Landfield P W.
Corticosteroid modulation of hippocampal potentials: increased
effect with aging. Science 245:1505-1509, 1989. Kirkwood A, Silva
A, Bear M F. Age-dependent decrease of synaptic plasticity in the
neocortex of .alpha.CaMKII mutant mice. Proc Natl Acad Sci
94:3380-3383, 1997. Kuhl D, Skehel P. Dendritic localization of
mRNAs. Curr Opin Neurobiol 8:600-606, 1998. Landfield P W, Thibault
O, Mazzanti M L, Porter N M, Kerr D S. Mechanisms of neuronal death
in brain aging and Alzheimer's Disease: Role of endocrine-mediated
calcium dyshomeostasis. J Neurobiol 23:1247-1260, 1992. McKay J A,
Murray G I, Keith W N, McLeod H L. Amplification of fluorescent in
situ hybridisation signals in formalin fixed paraffin wax embedded
sections of colon tumour using biotinylated tyramide. Mol Pathol
50:322-5,1997. Mackler S A, Brooks B P, Eberwine J H.
Stimulus-induced coordinate changes in mRNA abundance in single
postsynaptic hippocampal CA1 neurons. Neuron 9:539-548, 1992.
Mackler S A, Eberwine J H Diversity of glutamate receptor subunit
mRNA expression within live hippocampal CA1 neurons Mol Pharm
44:308-315,1993. Monyer H, Jonas P. Polymerase chain reaction
analysis of ion channel expression in single neurons of brain
slices. In: Sakmann B, Neher E. (Eds.) Single Channel Recording,
New York, Plenum Press, pp. 357-373, 1995. Monyer H, Lambolez B.
Molecular biology and physiology at the single-cell level. Curr
Opin Neurobiol 5:382-387, 1995. Nair S M, Werkman T R, Craig J,
Finnell R, Joels M, Eberwine J H. Corticosteroid regulation of ion
channel conductances and mRNA levels in individual hippocampal CA1
neurons. J Neurosi 18:2685-2696, 1998. Paradies M A, Steward O.
Multiple subcellular mRNA distribution patterns in neurons: a
nonisotopic in situ hybridization analysis. J Neurobiol 33:473-493,
1997. Perez-Reyes E, Wei X Y, Castellano A, Birnbaumer L. Molecular
diversity of L-type calcium channels. Evidence for alternative
splicing of the transcripts of three non-allelic genes. J Biol Chem
265:20430-6, 1990. Porter N M, Angelotti T P, Twyman R E, Macdonald
R L. Kineticproperties of .alpha..sub.1.beta..sub.1 -aminobutyric
acid.sub.A receptor channels expressed in chinese hamster ovary
cells: regulation by pentobarbital and picrotoxin. Mol Pharmacol
42:872-881, 1992. Porter N M, Thibault O, Thibault V, Chen K C,
Landfield P W. Calcium channel density and hippocampal cell death
with age in long-term culture. J Neurosci 17: 5629-39, 1997. Raap A
K. Advances in fluorescence in situ hybridization. Mutat Res
400:287-98, 1998. Ramsay G. DNA chips: state-of-the art. Nat
Biotechnol 16:40-44,1998. Schena M, Shalon D, Davis R W, Brown P O.
Quantitative monitoring of gene expression patterns with a
complementary DNA microarray. Science 270:467-470,1995. Schena M,
Shalon D, Heller R, Chai A, Brown P O, Davis R W. Parallel human
genome analysis: microarray-based expression monitoring of 1000
genes. Proc Natl Acad Sci 93:10614-10619, 1996. Speel E J, Hopman A
H, Komminoth P. Amplification methods to increase the sensitivity
of in situ hybridization: play card(s). J Histochem Cytochem
47:281-8,1999. Speel E J, Saremaslani P, Roth J, Hopman A H,
Komminoth, P. Improved mRNA in situ hybridization on
formaldehyde-fixed and paraffin-embedded tissue using signal
amplification with different haptenized tyramides. Histochem Cell
Biol 110: 571-7,1998. Sudweeks S, Twyman R E. Single cell reverse
transcription-polymerase chain reaction (RT-PCR) and the gabaa
receptor. Neurochem Int 2:137-139, 1996. Tank D W, Sugimori M,
Connor J A, Llinas R R. Spatially resolved calcium dynamics of
mammalian purkinje cells in cerebellar slices. Science 242:773-777,
1988. Thibault O, Mazzanti M L, Blalock E M, Porter N M, Landfield
P W. Single-channel and whole-cell studies of calcium currents in
young and aged rat hippocampal slice neurons. J Neurosci Meth
59:77-83, 1995a. Thibault O, Porter N M, Mazzanti-Rose M L,
Campbell L W, Blalock E M, Landfield P W. Dual patch pipefte
recordings in hippocampal neurons: evidence that long Ca.sup.2+
tail currents reflect Ca.sup.2+ channel activity at resting
potential. Soc Neurosci Abs 21:1577, 1995b. Thibault O, Landfield P
W. Increase in single L-type calcium channels in hippocampal
neurons during aging. Science 272:1017-20,1996. Thibault O, Porter
N M, Chen K-C, Blalock E M, Kaminker P G, Clodfelter G V, Brewer L
D, Landfield P W. Calcium dysregulation in neuronal aging and
Alzheimer's disease: History and new directions. Cell Calcium
24:417-433, 1998a. Thibault O, Clodfelter G V, Landfield P W.
Ca.sup.2+ transients during synaptic activation in hippocampal
sliceneurons of adult and aged rats. Soc Neurosci Abs 24:1971,
1998b. Tkatch T, Baranauskas G, Surmeier D J. Single cell RT-PCR
demonstrates a linear relationship between a-like current and KV4.2
expression in striatal and pallidal cell types. Soc Neurosci Abst
24:1330, 1998. Van Gelder R N, Von Zastrow M E, Yool A, Dement W C,
Barchas J D, Eberwine J H. Amplified RNA synthesized from limited
quantities of heterogeneous cDNA. Proc Natl Acad Sci 87:1553-1667,
1990. Watson A, Mazumder A, Stewart M, Balasubramanian S.
Technology for microarray analysis of gene expression. Curr Opin
Biotechnol 9:609-614,1998. Winder D G, Mansuy I M, Osman M, Moallem
T M, Kandel E R. Genetic and pharmacological evidence for a novel,
intermediate phase of long-term potentiation suppressed by
calcineurin. Cell 92:25-37, 1998.
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